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I have a western blot mystery.
I see protein on my ponceau stain, my beta actin is relatively even, but two of my histone three lanes are basically missing signal... This is not the first time this has happened and I'm very unsure how ponceau could show signal but not when probed for H3....
Technical specs
  • 8e6 cells (OCI-AML3) harvested, lanes are WTbulk, Clone 1, clone 2, clone 3
  • Lysed in 80ul of RIPA (commercial) + Phosphatase/protease inhibitors, 30 mins on ice with vortexing every 10 minutes
  • BCA showed no oddities
  • Pre-cast gel run with 40ug of protein, 80V for 1.5 hours
  • Transfer with biorad turbo transfer
  • Blot dried for 1 hr
  • Reactivated and stained with ponceau (see image attached)
  • Blocked for 1hr with commercial blocking buffer
  • HRP linked beta actin (1:1500), total H3 (m)(1:1000), and H3K9me1 (r) (1:1000) used to probe with HRP secondaries at 1:1500
Any advice or suggestions would be greatly appreciated!
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yes , this is weird . if your sample do not need to be in a native lysis buffer (ie: for use in IP expt) . try lysis in a fully denaturing buffer 1%SDS (with out detergent, and sonicate)
. and quantitated with ...Does SDS affect the BCA assay?
Note: 5% SDS is compatible with the BCA Protein Assay.
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Hello everyone,
I recently cloned EGFP into pET-28a(+) and transformed it into BL21(DE3) cells. However, despite trying multiple conditions, I am not observing any yellow-green color or fluorescence under UV light. Here’s what I’ve done so far:
  • I did Ligation 1:7
  • Colony Selection: I picked colonies via colony PCR to ensure the insert was present.
  • Induction Conditions: I tested 0.1–0.5 mM IPTG at both 37°C
  • UV Inspection: I checked fluorescence using a standard 366 nm UV lamp, a UV transilluminator, and a Bio-Rad longwave UV lamp but saw no significant fluorescence.
  • Agar Plate Test: I streaked 8 new colonies on an 0.1mM IPTG-supplemented agar plate, yet none of them showed fluorescence under UV.
  • Liquid Culture Observation: The culture itself appears slightly greenish, but when I pellet the cells, there is no visible fluorescence in the pellet.
  • Frame Shift Check: I verified my cloning strategy, and there is no frame shift issue.
To give some visual context, in the attached photo:
  • The left flask contains culture induced with 0.2% lactose( not necessery I just tried something)
  • The top-right flask (faintly green) was induced with IPTG in LB.
  • The bright green flask on the right was induced with 1% lactose ( not necessery I just tried something)
  • Falcon is LB medium.
Any insights or suggestions would be greatly appreciated! Thanks in advance.
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Kadir, thank you for the additional information. You absolutely need to know the sequences of the vectors encoding 1) EGFP, 2) pET-28a [you seem to have this one], and 3) the final cloned product. The sequences of the first two must be known BEFORE you start cloning and the last one must be confirmed AFTER cloning and BEFORE proceeding to protein overexpression. This is standard protocol otherwise you take a huge risk wasting months worth of time— believe me it can happen I wasted 6 months on working with the incorrect clone. I think it would be best for you to start cloning afresh after getting the proper information. Ask for the sequence of the plasmid encoding EGFP that is being used as the template for PCR and ensure that its sequence is known and how it was confirmed. If you obtained pET-28a from Novagen then it is probably fine, but please check. Once you have the in silico files of each, make a new in silico file of pET-28a encoding EGFP (the product you expect following successful cloning) and then add the primers to each relevant file. If you wish, you can send me a private message to set up a Zoom call, that would be much easier than sending messages back to back. Please let me know if you have further questions.
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They are charging INR 2700 per paper. The UGC CARE portal indicates that it is a cloned or hijacked journal. Any lead would be helpful.
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Dear Soma dey Sarkar JUSST stands for the “Journal of University of Shanghai for Science and Technology” and the Chinese title “Shanghai Ligong Daxue Xuebao” Indeed there is a hijacked/cloned version of this journal.
According to the “Retraction Watch Hijacked Journals Checker” https://retractionwatch.com/the-retraction-watch-hijacked-journal-checker/ the following website is the hijacked version https://jusst.org According to SCImago the real homepage is https://jns.usst.edu.cn/shlgdxxbzk/home
Indeed this information can also be found in the UGC-CARE List of cloned journals Group II https://ugccare.unipune.ac.in/Apps1/User/Web/CloneJournalsGroupIINew
Even if you missed all this vital info there are numerous red flags which can be identified for the fake version:
-No real contact info https://jusst.org/contact-us/
-The journal title clearly indicate that its origin is Chinese and more specific Shanghai, no one of the editorial board has any relation to this origin https://jusst.org/editorial-team/
-If one scroll down to the indexing info https://jusst.org/ it claims a DOAJ membership while they are not
-Papers have a non-functioning DOI
-The number of papers mentioned here https://jusst.org/archive/ do not match with the steady 70-80 papers annually which can be seen here https://www.scopus.com/sourceid/14040 (click on Scopus content coverage)
-If one clicks on processing charges here https://jusst.org/call-for-papers/ one gets an error (with other words: they hide the real costs)
For the most complete and updated list of identified hijacked journals I recommend “Retraction Watch Hijacked Journals Checker”
Best regards.
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Hello All,
I'm trying to clone a 1.5 kb fungal gene to a PJET cloning vector (Thermo scientific) that has 98% selection capability, and I'm also verifying the clone using the PCR, but every time after sequencing, I get the result as a vector sequence rather than the specific clone fragment that I'm targeting. Please suggest me if any one aware about this problem?
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Hello Sumit, more information would help us troubleshoot. Can you kindly attach your protocol? Alternatively , you can try RecA-independent recombination or RAIR cloning, see publication below. You just need four primers—two to amplify the vector and two to amplify the insert— where the 5’ and 3’ ends (of the resulting PCR amplicons) are homologous to one another. Following PCR, you just mix 1-2 microliters of each reaction to heat shock cells, perform the heat shock protocol, plate the cells, and perform colony PCR the next day to select the positive clones. Most common laboratory strains of E. coli are capable of RAIR. Good luck!
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I want to clone my 60 bp insert into my approximately 7000 bp vector. I did the ligation with a vector to insert ratio of 1:10. Either very few or no colonies grew. Also my colony PCR results were negative. How should I change the vector insert ratio?
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Hello Elif, I agree with Eddy— we need more information to provide useful feedback. Sounds like your competent cells or protocol for transformation may be part of the issue. Have others in your lab had issues with those cells? Could you attach the protocol you used? Honestly, your insert seems small enough that you may just be able to order two primers to do your cloning. For example, each primer could be 45 nucleotides long (just making a number up) where 15 anneal to the vector and the other 30 are part of your insert. Following PCR, just do a DpnI digestion, PCR cleanup, ligation, and then transform your cells. You would need to account for melting temperatures, secondary structures, etc. given the length of the primers, but just an idea. You could also try Rec-A independent recombination or RAIR cloning, see the publication below. Most common laboratory strains of E. coli are capable of RAIR. All you would need to do is buy four primers, two to amplify the vector and two to amplify the insert, with the 5’ and 3’ ends being homologous to one another. After PCR you just add 1-2 microliters of each reaction to heat shock cells, carry out heat shock protocol, plate the cells, and do colony PCR the next day to screen for successful clones. Good luck!
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I have been struggling with getting clones with LR plus reaaction to clone three fragments into a destination vector. I use 5fmol of entry clones and 10fmol of the destination vector. I first calculate the fmol : (concentraion in ng x 10^6)/ (size in bp x660). I then calculate and dilute the sample and keep the reaction. for eg: if i get 29.9 fmol in concentration for an entry clone and to bring it to 5fmol, i would take 1.67uL of the plasmid and dilute in 8.33uL water and from it take 1uL for the reaction. So, I am taking 1uL of all three entry clones (of 5fmol each), 1uL of destination vector (10fmol) and 1uL of LR plus clonase. Can anyone suggest what I am doing wrong?
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Gayathri, your protocol is consistent with the Invitrogen protocol so unsure if concentration is the issue. A few questions. How exactly are you quantifying the vectors? Are you transforming into commercial or home-made heat shock cells and are they the same One Shot cells? The invitrogen manual says, “We recommend using plasmid DNA purified with the PureLink™ HiPure Plasmid Midiprep Kit. Mini-prep (alkaline lysis) DNA preparations are not recommended for MultiSite Gateway™ Pro cloning reactions. DNA cannot be quantitated by UV absorbance because of contaminating RNA and nucleotides.” How are your plasmids being isolated? You can linearize your vectors using either PCR or restriction enzyme digestion. The former may be more desirable because you can then DpnI digest the original supercoiled vectors that would be preferentially taken up by cells. The smaller, original vector will always be preferentially taken up by the cells compared to a larger vector (generated by LR clonase). I also assume that the LR clonase reaction is not complete and there are still the original vectors that have not undergone recombination. Have others in your lab tried LR cloning and have they had these issues? Thanks!
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I have tried In-fusion cloning few times and it used to work in my hand. Suddenly, after few months when I again wanted to do some more cloning, it is not working as in I am not getting any clones in the test plates.
I have used gel purified vector (fully double digested as observed in gel) and gel purified PCR products. The homology overlap of the PCR products were 15 bp.
I have used 50ng of vector and the Vector : Insert molar ratio was 1:4. The In-Fusion reaction incubation was done in a water bath set at 50C for 1 hr. It would be of help if I can get any advice regarding this.
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Always try controls given in the kit. If they dont work something is wrong with the master mix
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Hi, it's been over a month that I am struggling with my clones. I got the desired clones for my study from some lab. They gave me the clones in the form of master plate. Now when I replated it and also made glycerol stocks from it with proper antibiotics, I did a colony PCR to check my construct is there in it or not. I pick up one single colony and put PCR and when I get the colony positive I put the same colony for restreaking on a plate and then use it the next time for my study. Now what's happening is no matter how many times I screen colonies sometimes I get a positive colony PCR sometimes not. What could be the possible reason? I am getting colonies even after doubling the antibiotic concentrations. I am suspecting that the colonies have a mixed population of cells having transformed ( non recombinant) and transformed (recombinant). Anybody who can help?
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Generally it's not easy to extract plasmid from agrobacterium.any suggestions would be of help
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I want to clone a 67 bp insert into the prmc2 vector. I have annealed primers with RE site overhangs. I got false colonies without dephosphorylation of the vector and no colonies after dephosphorylation of the vector and phosphorylation of the insert. I also used different concentration for ligation reaction with different incubation time still get no result
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If i undertand well. you amplified the insert adding the restriction site regions and did you performed a standard ligase? Is it correct?
Which site did you used?
However as general comment, to insert so small region you can avoid to amplify and clone an insert but you can just perform the plasmid PCR using a stratregy similar to single point mutagenesis. You have to add in the primers a region with codify for the region to be inserted (with overhangs) using the PIPE cloning strategy
you can find an example of it at the following link:
good luck
Manuele
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Hi, I have been trying to clone topoisomerase-3B from C elegans cDNA in NEB 5-alpha Competent E. coli. I performed hifi assembly of the top-3B cDNA insert and RE digested yeast two-hybrid pGBD vector and transformed it in e. coli. However, none of my transformed colonies on the selection plates have the plasmid with top-3Binsert. Does anyone have any suggestions for cloning and transforming top-3B in E. coli?
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TOPO vectors are just for cloning, they are not expression vectors. You move into a different vector to induce protein expression.
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Im working as a lab technician at an educational facility in Montréal, QC. Canada. We're already cloning plasmid and DNA using bacteria but to be honest restriction enzyme are kind of expensive !! Since students tend to make mistakes using expensive enzyme, I'm really looking forward to have the expertise to synthesize our own. I need guidance please.
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it would be difficult if you insert it in a plasmid, because the enzyme would cut the genome and also the plasmid once it is produced. bacteria that produce restriction enzymes cover the restriction sites in the genome with methyl groups in order to avoid self-destruction.
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Hi everyone,
I want to buy a primary anti-PSMA (prostate specific membrane antigen) antibody for western blot and immunofluorescence purposes. I am considering to acquire one of these three:
- Abcam ref: ab76104 clone: EP3253
- Cell Signaling Technology ref: 12702 clone: D4S1F
- Invitrogen ref: 37-3900 clone: 1H8H5
Have any of you used any of these antibodies? If so, I would really appreciate some feedback :)
Or if you use a different anti-PSMA antibody besides these and can recommend it please let me know as well!
Thank you so much,
Diana
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Hello Everyone,
I have recently been inquiring about recommendations for the optimal anti-PSMA (prostate-specific membrane antigen) antibody suitable for Western blot and immunofluorescence applications. If any of you have experience using these antibodies or if you can suggest an alternative anti-PSMA antibody, I would sincerely appreciate your feedback and insights. Your expertise and recommendations are crucial in assisting me in identifying the most effective antibody for my research objectives.
I would like to inquire if the previous recommendation continues to be effective?
Thank you for your assistance.
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Hello,
I am currently working on a cloning a 6 kb insert into 14 kb vector by Gibson Assembly. Following heat shock transformation into chemically competent TOP10 cells, I had multiple positive colonies as identified by colony PCR (1 vector-specific + 1 insert-specific primer).
However, following miniprep all colonies were found to contain the original 16 kb vector (all plasmids were run on a gel as a 6 kb insert difference should be easily distinguishable from the 14 kb vector + two colonies were sequenced by whole plasmid sequencing to verify). All fragments are amplified by PCR, and I use DpnI digest + small template amounts (1 ng) to minimize template carryover. I also use a blank plate which substitutes the insert for an equal volume of water. The blank plate had only two colonies compared to 12 on the transformation plate. This cloning procedure has also worked successfully for me in the past with smaller fragments (<15 kb total construct size).
I am suspecting that the cells were unable to take up the 20 kb plasmid, and colony PCR was detecting the successful Gibson construct which was spread on the plate during transformation. I emailed ThermoFisher and they said 20 kb in TOP10 by heat shock should be fine, but I am wondering if anyone else can share their experiences. If chemically competent TOP10 is not feasible, should I think about electroporation or using an alternative strain?
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Hi,
yes, the transformation efficiency decreases with plasmid size.
First of all, you may try electroporation
Then you may use slightly slower grow condotions, likely 25...30C.
You may try rising Mg2+ and/or Mn2+ in your heat shock transformation buffer.
You may use positively charged peptides (like lactoferricin B or so) to aid plasmid with neutralized surface charge to enter the cell (there are som works on cell penetrating peptides, but I don't know whether synthesis or purchase is an option for you). In this case you may first mix plasmid and a peptide in mQ at different ratios, then add 2...3volumes of bacterial suspension and leave at 20...25C for 10...60min, then spin, resuspend pellet, plate.
Of course, in all the cases there will be a need for titrations etc.
Sorry, no magic bullet.
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There is an organism Z that contains many putative protease genes. I want to clone some of them, so I need bioinformatics support to finalize the rationale for choosing these specific enzymes. So can anyone help me in this.
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To study new features in proteases and peptidases that might make them work better, we can use several bioinformatics methods. First, we look at their sequences to find patterns or regions that are similar to known enzymes using tools like Pfam or InterPro. If there are new regions, we compare them across multiple sequences to find important parts. Next, we predict their 3D shapes using tools like AlphaFold to see how they might work. We can also compare them to similar enzymes to spot differences. Finally, by studying their evolution and using computer models, we can predict which features make them special and test these ideas in experiments.
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Hello,
I'm doing site directed mutagenesis. So far, I didn't have any issues. For one construct only, I get insertions that are equal to my primer sequences. Sometimes it is 1, sometimes it's 3 insertions, depending on the clone. I know that this can sometimes happen when the transformed bacteria ligate the nicked ends of the plasmid. But in my case, I did an agarose gel extraction of my plasmid before transformation. I cannot really explain where those primer inserts come from. I assume it must happen during PCR? Is there any way to avoid this phenomenon?
Thank you very much.
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Well, any chance your primers have complementary ends that allows them easily stick end to end?
You could lower the primer concentration in your PCR, that should help a bit.
If you are still getting your desired construct often enough to work then don't spend too much time tracking down the cause.
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I am wondering what is the maximal distance the U6 promoter can be situated from its target gene and still drive expression? I am developing a model whereby U6 will be located approx. 160 bp away from the gene of interest (last base of U6 promoter to 1st base of target gene sequence). Should this still drive expression? Is there any literature that people are aware of on the topic? I was intending to test this in a plasmid model first but the cloning is being awkward. Any guidance appreciated.
Thanks in advance
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Hi Dylan,
The U6 promoter is typically used for short RNA expression (gRNAs, shRNA etc) but I don't know of any example for protein coding genes. Depending on your application it might or might not be the right choice.
The transcription will start inevitably from the first "G" after the promoter, and it will stop at the first short stretch of poly-T (usually 5). 160 bp is quite a long gap for this promoter, and there's a risk of appending significant portions of RNA at the 5' end of your gene or, if a poly T occurs, stopping tanscription prematurely.
I would try to avoid such a big gap.
Hope it helps
Francesco
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I am planning to clone an enzyme using the pET28a vector. I would like to ask whether the N-terminal His-tag alone would be sufficient for efficient expression and purification, or if it might cause the protein to misfold, making it difficult to purify.
Would it be more effective to place the His-tag at the C-terminal of the enzyme instead? In this case, I would add a stop codon in the reverse primer.
Which approach would be better for ensuring proper folding, expression, and successful purification of the enzyme?
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Check the Alphafold predicted structure of your protein. If the N-terimus is deeply buried in the protein or involved in protein-protein interactions, try the C-terminus. Probably best to do both though since it isn't that much extra work.
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I have been using NEB Hifi Gibson assembly for a couple years now and I've been quite happy with it. I regularly make plasmid constructs with 4-8 fragments, and always >1/4 of the colonies are "perfect," while the remaining ones may have some SNPs at the joining sites, or be misassembled due to a repetitive region.
Some colleagues said that Golden Gate has even higher efficiency. That even with 8 fragments, it is normal for 50-100% of colonies to be perfect. Is that true? Is Golden Gate that perfect?
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Has anyone tried GenScript's GenBuilder DNA assembly kits?
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Hi,
I am having troubles cloning a Cas9 gene (around 4300 kb) into a vector, replacing a LacZ cassette. (I use the uloop system.) I use SapI as a restriction enzyme and T4 as a ligase. I cloned several other constructs into the same kind of vector with high efficiency (mainly white colonies, and almost all carry the correct insert when screened).
When I try to insert Cas9 (or also dCas9) I yield the same number of colonies, with again almost all white. However, when I screen the colonies via colony PCR or restriction digest the isolated plasmids of the colonies, no vector carries the insert. I did not sequence these plasmids, but from the restriction digest it suggest, that the plasmid only lost its LacZ cassette and was closed again without any insert (eventhough overhangs are not compatible).
I tried two settings for GGC:
37 C 5min...............37 C 5min
16 C 5min................16 C 5min
(25cycles)................(40cycles)
65 20min-................65 20min
85 10min..................85 10min
4 hold......................4 hold
I tried two different buffer conditions:
-T4 buffer only
-Or 50% T4 buffer, 50% Cutsmart buffer
I started with fresh Cas9 (and dCas9) PCR templates and with several fresh receiver plasmids. Also I used different competent cell batches.
I double checked the overhangs of receiver plasmids and the overhangs created on the Cas9 insert.
I am running out of ideas..
Does anyone know if GGC with single large inserts (4000 kb+) effect GGC efficiency?
Does anyone have a suggestion how I could improve GGC efficiency?
Further troubleshooting suggestions?
I really would appreciate your ideas!
Thanks,
Florian
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Dear Florian ,I was wondering if you could manage the problem because I have the same problem with Golden gate cloning for a 5kb insert.
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Does anyone have experience with the Anza™ Restriction Enzyme Cloning System? Have you ever had any problems?
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I think we did. The most important issue was the heat inactivation of some enzymes - it seemed like they didn't deactivate and a clean-up kit had to be used to get any results. The DNA amount for the reaction had to be closer to the upper limit (1ug) and the clear buffer was the only one working. After the ligation we used the highly competent dh10B cells (chemocompetent). For electrocompetent cells cleaning the DNA after the ligation was necessary but the majority of it was lost and the transformation efficiency was low.
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Is this toolkit only an extension for YTK (MoClo Yeast toolkit), does it assume that I have this kit and is based on YTK level 0 fragments?
Are there any regular YTK promoters or only the newly added inducible promoters?
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I think, newly added inducible promoter
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I am having an issue trying to clone 1.5 and 2Kb fragments into a lentiviral vector. I successfully cloned a different insert (1Kb) into the same vector on the same day. There were a lot of colonies on the blank (cut vector + ligase with no insert), but about half of the ligated colonies were correct. My problem inserts were cut and ligated at the same time, but came out of the incubator with no colonies (none on the blank either). (A different intact plasmid was also transformed at the same time and looked fine with lots of colonies.)
The only differences between the ligations was one restriction enzyme I used (PspXI + EcoRI worked, but PspXI + NotI did not work), and the DNA itself.
Does anyone have any thoughts? I got advice that some restriction enzymes "over cut" and reducing digest time helps sometimes, but reducing digest time to 5 min did not give colonies either.
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Have you checked the expiration date on the enzymes?
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I was trying to clone an RCA product (digested with a single restriction endonuclease) into the pUC 19 vector but I failed. After cloning, I have to go for the sequencing. Can I clone it into a TA vector?
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Alexandra Johnson yes, It should be a 2.7 kb begomovirus genome, which I have to confirm by the sequencing. The sequence is not known, so I can't amplify with PCR.
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After 30 years of having performed hundreds of CFAs using a large number of different mammalian tumor cell lines, the CFAs don't work any longer. We used to seed 300-500 cells per well into a 6-well plate and always had >80% plating efficiency. Now the cloning efficiency is 0% at these cell numbers. I am embarrassed that we can't figure out what's wrong.
The key problem is this: after seeding, the cells don't attach (and therefore don’t proliferate and form colonies). In comparison, when the cells are seeded at much higher cell densities, they attach perfectly well at >95% efficiency. We are working with standard monolayer tumor cell lines: MDA-MB-213; MCF7; B16; T98G; etc. The problem is the same with all cell lines we have tested (and used successfully in the past). For the past year, we have been unable to perform any CFAs with any of these cell lines.
There is an apparent threshold: when we seed any cell number from 100 - 5,000 cells per well (6-well), plating efficiency is <1% because the cells do not attach to the surface of the culture plate (at 24 hours after seeding, the cells remain round, some are semi-attached, many are floating, and they look sick). In contrast, when we seed >5,000 cells per well, cell attachment (plating efficiency) shoots up to >90% (at 24 hours after seeding, the cells are flattened and firmly attached to the surface of the culture plate, and they start proliferating). But of course, with >5,000 cells per well, you cannot get quantifiable colonies; after a week, you have a dense monolayer and CFAs are impossible.
Over the past year, we tried different plate formats (6-, 12-, 24-wells; 10-cm plates) from different vendors; used more FBS or conditioned medium; pre-coated with Matrigel; used trypsin-free cell collection before seeding; purchased DMEM from different vendors; used more/less medium in the wells; tested for mycoplasma. But we always get the same outcome with each cell line: once cell density during seeding is below about 500 cells per square centimeter, plating efficiency drops to near-zero; above that number, plating efficiency shoots up to close to 100%. Regrettably, under these conditions, CFAs are not possible. Any suggestions what else we could try?
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It seems your cells are struggling with adhesion at low densities. To troubleshoot, consider:
  1. Cell Condition and Handling: Verify passage numbers, reduce trypsinization time, and check for cellular stress (e.g., try antioxidants like NAC or lower oxygen conditions).
  2. Conditioned Media or Feeder Layers: Adding conditioned media from high-density cultures or using a feeder layer could support low-density attachment.
  3. Alternative Coatings: Try different coatings like fibronectin, collagen type I, or poly-D-lysine, which might enhance attachment.
  4. Growth Factor Supplementation: Consider adding ROCK inhibitors, EGF, bFGF, or IGF-1 to aid attachment.
  5. Cell-Cell Signaling: Test if adding supernatant from high-density cultures improves attachment, as low-density cells might lack autocrine support.
  6. Plate Surface: Use plates from various suppliers to rule out surface property differences.
  7. Low-Density Plating Protocols: Experiment with initial serum-free media, gentle rocking, or shaking to improve cell attachment.
  8. Contaminants: Ensure there are no subtle contaminants in the media, plastics, or reagents.
Testing a combination of these suggestions may help restore cloning efficiency in your CFAs.
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We utilize the pET22b vector for cloning and aim to incorporate the pelB signal sequence for periplasmic expression. For this purpose, we are using the restriction enzymes NcoI and XhoI. However, we have encountered a frameshift during translation due to NcoI. Does anyone have suggestions on how to resolve this issue?
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as Katie A S Burnette suggest probably you need to redesign your primers.
Ncoi due to the presence of an ATG codon.
CCATGG
is kwon to induce frameshift when you use it at the N-term.
To avoid this problem you need to add 2 extra basis before your GOI sequence that willl result in the addition of an extra AA at the N-term of your protein construct just after the methionine.
you can see an example of it at minute 3'00 of the following video
However as general rule i suggest you to leave the standard cloning based on restriction enzime and ligases and learn the PIPE cloning which is a powerfull enzime free cloning that make you independet from the presence of restriction enzimes in your vector
you can find more information about it on the following links
good luck
Manuele
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I am trying to clone my gene of interest in pENTER, size of gene is 2.5 kb and vector is also about 2.5kb. after many trials I am getting clone in reverse orientation. I have added CACC site on 5'end of forward primer. Kindly suggest me what could be the possible reason for this.
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How many colonies did you check?
If it's less than 20, then just check a few more.
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My PI wants to use expression plasmid for E. coli with two cloning sites. We have pColA-Duet-1 and pACYC-Duet, but in those are both of the sites inducible by IPTG (if I understood it correctly). Are there any plasmids that would have two different inducible promoters?
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Answering your question, as far as I know there is no such a vector , but you can easily modify/engineer the vector pACYS-Duet1 by replacing one of the IPTG-inducible promoters with the tet-inducible promoter from e.g. pASG-IBA2.
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hey,
im designing an experiment to optimise prime and base editing epeg/sg RNAs based on their efficiency and off target effects. I understand that using a cell line easy to transfect such as HEKs are ideal for this, and then select the best rna and use in your target cell. But I will need to insert the desired mutations in the HEK cells first… I thought doing the reverse prime/base editing was a bit of a long procedure just to correct the mutation again, so wanted to transduce them with a lenti carrying the gene with the mutation. This gene however does not fit into a lentivirus since it’s quite big, would inserting just a region of the gene that contains the mutations along homology arms (of1kb or how long?) be enough to validate genomic edit? ofc I wouldn’t be able to validate anything at the protein level but just to assess the efficiency at the gene level. has anyone done this or know of a publication which has? Or any reference that might suggest it’s a valid experiment? What do you think? thanks!!!
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There is no point. Why introduce cell type and that vector situation as new variables? This is not going to tell you anything about what happens in your final intended system. Optimize for what you want, as directly as possible.
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Dear all,
I have been trying to gateway clone two genes, both around 3.2kb in size. However, I am running into a peculiar issue where my colony PCR is positive but once I isolate the plasmid, the insert is not there. For both genes this issue is coming up after the BP reaction transformation. I am using pDONR221. I have also set up a control PCR with my gene primers and pDONR to see if there was any non specific binding but that is not the case. I had previously cloned a 1.2kb gene in the same vector so I am wondering if this is an issue due to the size of the genes.
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Nope.
Colony PCR is notorious for false positives, especially if you are picking from the selection plate. If you insist on doing colony PCR, you need to streak out colonies of interest on a new plate & start a liquid culture from that streak plate.
I'd make a streak plate, set up a liquid culture and do a proper plasmid prep. As my advisor said, "You can't rush quality".
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Dear cloning community,
Is it possible to use Overhangs with large Tm difference with NEB Builder HiFi DNA assembly?
5´ 26 bp Tm 57ºC
3´ 24 bp Tm 72ºC
Unfortunately, extending the 5´ doesn't help much because it is a high TA region, and the same goes for reducing the 3´(High GC region)
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Hi, thank you for the answers. The high CG content was unavoidable as it was part of the MCS of the plasmid into which we needed to insert it. In the end I prolonged the arms and they had 30 -38 bp depending of the arm, and Tm of 60 till 79ºC and it worked. Just leaving this info here in case someone has the same question in the future
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I'm trying to clone some large pieces of cyanobactrial gDNA (sometimes even more of them) into a plasmid via fusion PCR. Then, I transform it into E. coli TOP10, as my PI says it works better (for HiFi DNA assembly).
However, I quite high percentage of my positive clones had some problems in the plasmid elsewhere (like missing I think 2.3 kbp of the plasmid; or some mess in another case etc.).
It is true, that when I compared TOP10 and DH5alpha, TOP10 had many more colonies (more than 10-times). However, I think that DH5alpha should be less prone to DNA alterations, right? So should I use rather DH5alpha and hope that I will get at least few colonies, but they will be positive?
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I have tried both methods. Sometimes PCR cleanup is enough and gives good results but if the fusion is problematic, gel purification is much better.
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15 E. coli K12 promoters from EcoCyc are now accessible on PeptiCloud (www.pepticloud.com)! You can clone them into your projects to build constructs. Find them here: https://www.pepticloud.com/public-project/E.%20coli%20K12%20Promoters.
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Thanks for sharing
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Hello everybody,
I'm developing some truncated versions of ROR1 for in vitro studies and I would like to check by flow the absence of signal when targeting ROR1. I know that 2A2 binds to the N-ter, and most likely this means the Ig-like domain, but I don't have any clue whether exists an antibody (commercially available) against Frizzle or Kringle domains.
Does anyone know what epitope is each of these clones binding to? Is there any webpage where I can get this info?
Thank you very much in advance for all your help.
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When developing antibodies against truncated versions of ROR1 and checking signal absence by flow cytometry, it is crucial to understand the domains of ROR1 and their corresponding antibodies.
ROR1 structure contains several functional domains:
- Ig-like domain: This is the N-terminal part of ROR1 and is usually associated with the reception of extracellular signals.
- Frizzled (Fz) domain: This domain is associated with the Wnt signaling pathway and is involved in intercellular signaling.
- Kringles domain: This domain is commonly found in various proteins and may be associated with extracellular matrix binding and cell migration.
Antibody binding site: According to existing research data, monoclonal antibodies (mAbs) against ROR1 mainly recognize epitopes in the following two regions:
1. Ig-like domain:
- Most antibodies, including 2A2, mainly recognize the Ig-like domain of ROR1. This indicates that the binding site of 2A2 with ROR1 is likely to be located in the N-terminal region, which is very important in flow cytometry because it can effectively label and detect the expression of ROR1.
2. Frizzled and Kringles domains:
- Regarding antibodies against the Frizzled domain, although there is no clear indication in the literature that there are specific antibodies, studies have shown that the Frizzled region of ROR1 also has binding ability with other antibodies such as R12, which may target epitopes in the Frizzled region [2][3].
In flow cytometry, antibodies against different domains of ROR1 can help us better understand the role of ROR1 in cell signaling. By comparing the binding properties of other antibodies, the importance of different domains of ROR1 in signaling can be evaluated. For example, the 2A2 antibody has been shown to effectively block Wnt5a-induced ROR1 signals, which further supports the key role of ROR1 in cell migration and proliferation [6][7].
- Regarding antibodies against the Kringel domain, there is no direct mention of specific antibodies against this domain in the current literature. Nevertheless, studies have shown that different domains of ROR1 may be recognized by different antibodies, which provides a possible direction for the future development of antibodies against the Kringel domain [1][4]. For example, existing anti-ROR1 antibodies show different binding properties and effects, which may be related to the specific epitopes they bind to[4][5].
References:
[1] T. Tran. Armored tgfβriidn ror1-car t cells reject solid tumors and resist suppression by constitutively-expressed and treatment-induced tgfβ1, Journal for Immunotherapy of Cancer, vol. 12, no. 4, p. e008261, 2024. https://doi.org/10.1136/jitc-2023-008261
[2] M. Hudecek, M. Stanghellini, , et al. Receptor affinity and extracellular domain modifications affect tumor recognition by ror1-specific chimeric antigen receptor t cells, Clinical Cancer Research, vol. 19, no. 12, p. 3153-3164, 2013. https://doi.org/10.1158/1078-0432.ccr-13-0330
[3] J. Qi, X. Li, et al. Potent and selective antitumor activity of a t cell-engaging bispecific antibody targeting a membrane-proximal epitope of ror1, Proceedings of the National Academy of Sciences, vol. 115, no. 24, 2018. https://doi.org/10.1073/pnas.1719905115
[4] R. Mani, Y. Mao, et al. Tumor antigen ror1 targeted drug delivery mediated selective leukemic but not normal b-cell cytotoxicity in chronic lymphocytic leukemia, Leukemia, vol. 29, no. 2, p. 346-355, 2014. https://doi.org/10.1038/leu.2014.199
[5] L. Aghebati‐Maleki, V. Younesi, et al. Antiproliferative and apoptotic effects of novel anti-ror1 single-chain antibodies in hematological malignancies, Slas Discovery, vol. 22, no. 4, p. 408-417, 2017. https://doi.org/10.1177/2472555216689659
[6] M. Hasan, L. Rassenti, G. Widhopf, J. Yu, & T. Kipps. Wnt5a causes ror1 to complex and activate cortactin to enhance migration of chronic lymphocytic leukemia cells, Leukemia, vol. 33, no. 3, p. 653-661, 2018. https://doi.org/10.1038/s41375-018-0306-7
[7] P. Janovská, L. Poppová, et al. Autocrine signaling by wnt-5a deregulates chemotaxis of leukemic cells and predicts clinical outcome in chronic lymphocytic leukemia, Clinical Cancer Research, vol. 22, no. 2, p. 459-469, 2016. https://doi.org/10.1158/1078-0432.ccr-15-0154
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Hello there
I have some difficulties in my cloning project, and I'd appreciate using your experience in this field.
I want to clone my insert (2.8 kb, cloned in pUC57) into a manually engineered plasmid for CRISPR (9 kb with pUC plasmids backbone) in XhoI restriction site.
For the experiment, I received my synthetic insert in pUC57, then, I prepared my insert with PCR (with speedy pfu as a polymerase with proofreading). (Notice: I can't prepare my insert with enzyme digestion and purification because my digested insert and linear plasmid have same size)
Additionally, I extracted my plasmid by Qiagen kit and eluted with nuclease free H2O. I used Xho1 treatment for both Insert (PCR product) and Plasmid in my digestion process, and I tried the gel purification for preparation of my XhoI digested-insert and eluted with nuclease free H2O. In order to do single digestion cloning, I needed to use alkaline phosphatase treatments (Roche) after plasmid precipitation and deactivate it in different ways for multiple cloning set up. So, the XhoI digested plasmid was treated with Alkaline phosphatase and deactivated in different ways including incubation at 65°C, gel purification, and clean up in independent cloning experiments.
For the ligation step, I tried 48h/4°C and 24h/16°C incubation and pre-warming of Insert+ plasmid in 45°C and 65°C, ice incubation, and then adding ligation buffer and ligase (Takara and Roche ligase were tested). I checked my ligase and it works well in different project.
Recently I tried my cloning process with double digestion by forward primer changing (replacing NcoI instead of XhoI) in preparing Insert by PCR, but I couldn't get any cloned plasmid.
I have tried different protocols, and I welcome your comments and experiences in this regard.
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1. You can solve your issue with the insert and plasmid being the same size by finding an enzyme that cut inside the plasmid but not the insert so the plasmid fragment will be broken to several pieces.
2. If you just cut the ends of a PCR product and linearizing a plasmid you can use PCR purification instead of gel extraction since the yield is usually better.
3. XhoI require at least 4 bases before the site to work efficiently, if you don't have that you can just add extra bases to your primer before the restriction site.
4. For phosphatase treatment, I just add it to the restriction reaction and let it sit for an additional hour or so, I don't bother with the inactivation since most of it will be gone in the purification step.
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I have to do cloning gene in pseudomonas, but we didn't a suitable vector. Does anyone know how can I design pucp18 from puc18 vector? Just, I add replication origin of pseudomonas, can I clone genes? and how can I do that?
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I am not an expert in this field, but I am very interested and have researched to find an answer. I received some assistance from tlooto.com for this response. Could you please review the response below to see if it is correct?
To design a pUCP18 vector from the pUC18 vector for cloning in Pseudomonas, incorporate a broad-host-range origin of replication such as oriV from plasmid RK2, ensuring compatibility with Pseudomonas species [2]. Use PCR to amplify the oriV region, then insert it into the pUC18 backbone via restriction enzyme digestion and ligation. Include antibiotic resistance markers suitable for selection in Pseudomonas [1]. Verify the construct through sequencing and test its functionality via transformation into Pseudomonas cells. This approach allows for stable replication and maintenance of the vector in Pseudomonas, facilitating successful gene cloning [3][4].
Reference
[1]
Matsusaki, H., Manji, S., Taguchi, K., Kato, M., Fukui, T., & Doi, Y. (1998). Cloning and Molecular Analysis of the Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyalkanoate) Biosynthesis Genes in Pseudomonas sp. Strain 61-3. Journal of Bacteriology, 180, 6459 - 6467.
[2]
Aakvik, T., Degnes, K., Dahlsrud, R., Schmidt, F., Dam, R., Yu, L., Völker, U., Ellingsen, T., & Valla, S. (2009). A plasmid RK2-based broad-host-range cloning vector useful for transfer of metagenomic libraries to a variety of bacterial species.. FEMS microbiology letters, 296 2, 149-58 .
[3]
Yen, K., Karl, M., Blatt, L., Simon, M., Winter, R. B., Fausset, P., HSIENGS., L., Harcourt, A., Chen, K. K., & Amgen (1991). Cloning and characterization of a Pseudomonas mendocina KR1 gene cluster encoding toluene-4-monooxygenase. Journal of Bacteriology, 173, 5315 - 5327.
[4]
Kimbara, K., Hashimoto, T., Fukuda, M., Koana, T., Takagi, M., Oishi, M., & Yano, K. (1989). Cloning and sequencing of two tandem genes involved in degradation of 2,3-dihydroxybiphenyl to benzoic acid in the polychlorinated biphenyl-degrading soil bacterium Pseudomonas sp. strain KKS102. Journal of Bacteriology, 171, 2740 - 2747.
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Plasmid PX330 contains our desired clone. After several plasmid extractions using new kits and running on an electrophoresis gel, no plasmid was observed, whereas the same plasmid with the same kit used about two months ago was extracted well. What could be the reason for the failure of extraction? We even used DMSO and heat shock to eliminate the possible secondary structures in the plasmid, but in the end, no plasmid was observed on the electrophoresis gel. What solutions are there to solve this problem?
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Have you checked the kit components to make sure none have degraded or expired?
Has anyone else in the lab tried using that kit recently? If yes, what results did they see?
I assume you remembered to add in the antibiotic selection during culture growth, but those can also degrade/expire with repeated freezing & thawing.
I'm sure you'll figure it out!
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Hello, I have a question, I am starting with GWAS analysis.
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GWAS is studied in a variable gene pool. But clones have similar genotype,aren't they.! Then why would you use GWAS for such clones.
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Hello,
I am doing recombinant cloning. for this purpose i have to cut plasmid and then gel purify the right size band for further procedure. but in my case, i am using TIANGEN gel extraction kit and after purification i m not getting sufficient amount of Plasmid. however i am using 2ug to 3ug concentration of plasmid for restriction digestion and band size are exactly right and i am cutting agarose neatly with less gel on it but still getting very low amount after purification. has anyone use TIANGEN kit before? and also is there any other way to purify desired band from gel?
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Most of these kits bind denatured dna on a membrane and then elute in water or dilute tris buffer. You can usually improve the yield of purified dna by heating the elution buffer to 70c and leave it on the membrane for twice as long as the recommended time quoted in the kit and if necessary do another elution to make sure that all of the plasmid has been released from the membrane
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Hi all,
DNA sequences have traditionally been shared through literature, often scattered or hidden in appendices, making it difficult for the research community to access and use modular sequences.
To enhance collaboration and accessibility, PeptiCloud has introduced a new feature that allows researchers to effortlessly clone and import DNA sequences from other projects into their own. Inspired by open-source collaboration in the software industry, this feature aims to make it easy and error-free for researchers to utilize and build upon sequences created by others.
Check out the cloning feature at www.pepticloud.com!
Thank you,
Chris
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Hi Dr. Akter. It's completely free!
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I am going back to recovering some BAC clones for gene targetting. But I cannot find the ordering source. Anyone can help ?
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thanks
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The question "What are the most common challenges in selecting an appropriate vector for cloning a large gene?" addresses the complexities involved in choosing the right vector for successfully cloning and expressing large genes in various host organisms.
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Selection of appropriate vector.
Look for High number of GC content.
Look for Restriction clevage sites.
check for open reading frame. framshit mutation during inserting in the vector.
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Hi all,
Currently, I am having difficulty with Gateway LR cloning. Actually with only one gene. For other genes, the technology works almost %100. However, when I tried to transfer HDAC2 into my destination vector (pFRT-TO-DEST), cloning fails and I don't get any colony. However, other genes I tried to clone at the same time worked well. Have you had similar problem with LR cloning? Where would it go wrong?
P.S. I use Invitrogen LR CLonase II. I follow the exactly same protocol written in the instruction. I also tried to extend reaction time.
Cheers,
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Mert Burak Ozturk I am also trying the Gateway clone and having trouble of getting positive colonies after LR. So far, it only yielded me false positives. After sequencing, my entry vectors are fine and using 10fmol for entry and 20fmol for destination vectors. Can you please comment if linearising the destination vector helped you finally? Or if anyone has any other input, that would be great!
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I have been generating stable cell clones (antibody production) using the ExpiCHO system + antibiotics selection. Although I have been able to select a few producing clones after months of culturing the stable pool, I find it interesting that only <20% of cells from the pool (sometimes 10%) are positive (antibody-producing cells), after passaging them without antibiotics.
What are you guys experiences in generating stable cell lines? Is this % reasonable for a pool selected by antiobiotics, after 3-5 months of subculturing? Should it drop when you remove the antibiotics?
Detailed answers would be lovely accepted :)
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I think the percentage is low in this case.
1. You can try to ascertain transfection efficiency by transfecting GFP as control.
2. you can start with a lower concentration of antibiotic and follow the two-phase selection strategy they recommend, to increase chances of selecting even the low copy number clones.
Subham.
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In many cloning protocols, researchers introduce an intermediate cloning step using a vector like pGEM-T before transferring the insert into the final destination vector. Could you elaborate on the rationale behind this approach and the specific advantages it offers, especially when considering the additional time and effort involved?
What are the specific advantages of this approach?
Thanks
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pGEM-T plasmids are optimized to be very simple to clone into - just add in your PCR product. The conjugated ligase will catalyze the reaction. No need to phosphatase treat the plasmid, no need to set up a restriction digest, etc. You can clone in 1 day (PCR in morning; set up selection plates that afternoon). This used to take a week (PCR, overnight digest, cleanup, phosphatase the vector, cleanup again, overnight ligations, etc.). Better living with science & technology!
Also, intermediate vectors like pGEM-T are an easy to make any changes to your target sequence (e.g. add a tag or change promotor). These plasmids have multi-cloning sites, known selectable markers, lots of valuable features. Easy to then digest & move to final vector.
Talk with your advisor about your specific project to plan out how to make your desired plasmid.
Good luck!
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I'm cloning a fragment of 3200 nts into plasmid. The cloning was successful, however, 02 amino acids were mutated. Now I want to fix these 02 aa by site-directed mutagenesis technique using original DNA plasmid as template for PCR. The PCR product contains 02 kinds of DNA which are in the same length (original template and newly synthesized product). PCR product will be treated by DpnI to digest all the original DNA. The remain PCR product (my target DNA, linearized structure) will be purified and performed in-fusion to circularized into plasmid, then transformed to E. coli for propagation. Plasmid will be extracted from the E. coli and confirmed by NGS. I repeat some experiments, unfortunately, it seems original DNA was still partly remain after DpnI or the site-directed mutagenesis reaction was not successful (I think) making new plasmid still identical to the original template.
Now I want to check whether my target amino acid is fixed or not before sending sample to NGS optimizing cost benefit.
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I concur that you only need to do regular sequencing not NGS and this should much much less expensive. However if your clone picked up two mutations during the cloning, why not just look for another clone? That might be easier than doing two rounds of Site directed mutagenesis.
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Hi, everyone. I want to transfer two genes in to the pseudomonas bacteria that isolated from the soil.
The bacteria that I want to use for cloning haven't been identified and not be sequenced, previously. How can I undrestand which promotor is the best and can be clone without any problem?
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There are many ways to approach this problem depending on the resources available to you. But I think you should first identify the organism you are working with. There are molecular markers which can help you do this like ITS or rDNA.
Once you have confirmed it is a Pseudomonas sp. you can search the literature for promoters and even expression plasmids.
Hope this helps
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Hello,
We found three packages of Illustra™ MicroSpin™ G-25 columns in the cabinet of an unused lab. They are very old but have never been opened. I have never used this kit before, and I couldn't fully understand what it is used for from my internet search. Is it just a simple DNA purification kit, or is it something more functional? I am interested in recombinant protein production. Can I purify my ligation product with this before cloning into bacteria, or can I purify my PCR product with this before ligation? In which scenarios is this kit indispensable?
Thank you.
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These are sephadex spin columns and work on the basis of size exclusion. So dna of more than 10 bases long will flow round the beads and elute early while small salts will have to flow through the pores of the beads and will elute much later. They can be used for dna purification from most smaller molecules and have been used to remove radioactive salts from end labelling of oligos with the labelled oligo eluting first off the column. The G25 is an indication of the size range that can be separated on these columns
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I am cloning an overexpression plasmid with my protein of interest tagged with mScarlet. After transforming my ligated product into DH5α bacteria and plating on LB agar, I noticed colonies with a pink tint to them. Miniprep pellets and even DNA elutions were also pink. The plasmid has been sequence verified and has the confirmed sequence. After looking online, I wanted to be sure this is not yeast contamination so the USER cloning was repeated. Again I saw both white and pink colonies and had pink pellets upon centrifugation. This issue has persisted across multiple batches of agar plates. Others in the lab have been doing transformations and have not come across this so it seems to be specific to my plasmid only. Could this be expression of mScarlet from my plasmid? This would be weird it is under the expression of mammalian promoter and none of my other mScarlet plasmids have had this issue. I've attached some photos for reference (arrows pointing to pink and white colonies).
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don't panic!! this is not unusual: I got the same observation with sfGFP, redGFP...construct it depends on the plasmid construct probably in some of them there is a cryptic bacterial promoter and this fluorescent protein are so powerfull that even a small expression will give coloured pellet. more over
yeast will not grow at 37°C overnigt in LB and if you can get a plasmid from your culture it is most probably E. coli...
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I am using a vector that has repeat sequences, and using Mach1 competent cells. I heard that if the cells are not stable cells, then we shouldn't grow them in 37 to avoid recombination to happen between the repeat sequences.
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Mach1 cells are very stable at 37°C, so you can grow them at 37°C. You can also use 30°C if you want, but there is no issue if you use 37°C to grow Mach1 cells. The vector: insert ratios of 1:3 and 1:5 work very effectively. You can use either one of them or both. Best wishes!
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I have already cloned ALP gene of L rhamnosus into pET22b+ vector. Cloning is already confirmed by restriction digestion and sequencing. Cloning is achieved in BL21(DE3), Codon+, Rosetta and Lemo cells. Different concentration of IPTG has been used starting from 0.1mM to 1M. Different temperatures like 37oC 18oC 16oC were used to achieve expression.Still there is no expression. Under simulation I have used Snapgene to construct vector and simulate expression. Everything was perfect but still there was no expression after Induction and SDS PAGE. Please do suggest.
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Have you sequenced your final construct to be sure everything is correct?
When you say there is no expression, do you mean there is no activity or no protein? Can you see protein by western blot? You need to distinguish between whether the enzyme is not active in E. coli or whether it truly is not expressed.
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I've been trying to clone my N-terminal inserts in the comercial pEGFP-N1 vector.
Initially, I cloned my N-terminal insert into a pGEMT-easy vector to ensure that the insert digestion was done correctly. The enzymes I use for digestion are NheI and BamHI. Then, the inserts are purified using an agarose gel and an agarose gel extraction kit.
The vector is digested with the same enzymes and also purified in the same way. For some reason, when quantifying the DNA, the 260/280 ratio gives very high values (reaching values of 6 or 14). Then, a 1:3 ligation of vector:insert is performed and bacteria are transformed. So far, there have been no positive clones.
Im really worried cause I've been stuck for a really long time with this cloning, and I dont know what to now.
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I think the high 260/280 ratio is telling you that there is something contaminating your DNA, possibly a reagent from the agarose gel purification. I think it best to run a small amount of your purified fragment on a gel to be sure you have intact DNA. Whatever this is might also be inhibiting the ligation since it is likely organic chemical.
However you can also try the cloning without gel purification. Since pGEM-T is Amp resistant and pEGFP-N1 is (I think) Kan resistant, you can select solely for the recipient vector alone. I frequently just digest both DNAs with the enzymes, run an aliquot on a gel to confirm they are well digested then set-up your ligation. Now the trick is how to you remove the background of vector that does not have the insert. There are many restriction sites in the polylinker that will be present in the intact plasmid but will now be deleted in the recombinant (just make sure they are not on your insert) such as PstI or EcoRI and then digest your ligated product with this enzyme to remove the background of vector alone. This is an approach that has often worked well for me.
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This question seeks to address the growing concern of cloned academic journals, which are fraudulent duplicates of legitimate publications. It aims to guide researchers, scholars, and academics on identifying distinguishing features between authentic and cloned journals. The focus is on understanding verification methods, such as examining the journal's website, checking ISSN numbers, reviewing the editorial board, and cross-referencing with trusted databases. The goal is to ensure researchers submit their work to genuine and reputable journals.
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I use the psip-403 vector that contains the erythromycin resistance gene for cloning, and I also tested several strains of Escherichia coli bacteria (top10f, dh5 alpha) for this purpose, and the cloning results were that both cloning DH5-a harboring erythromycin resistance gene and non-cloning DH5-a survive in erythromycin contained agar plate. Im sure that the cloning was successful and I tested and cultured different concentrations of the antibiotic erythromycin in the agar medium (1-0.2 g of antibiotic in 1 ml of ethanol for 50 ml culture medium). Also, I tested two different brands of antibiotics (molecule, sigma and pharmacy antibiotic tablet form) and used different solvents such as ethanol and dmso, but the results were still the same. If possible, please guide me in this matter.
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Ignore the answer from Mahdi, it's AI-generated nonsense.
Your antibiotic selection strength is not correct and you are using WAY too much solvent for the volume of media.
Here's a link to a lab website with instructors for how to make many of the common selection media types.
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Hi everyone,
I'm trying to obtain a heterozygous clone (wt/KO) using a CRISPR-Cas9GFP RNP approach on iPSC-Like. After the nucleofection, 90% of cells are GFP+. After sorting cells, only 4% have a wt allele. I did a clone selection to find a wt/KO but none have this genotype. Worse still, no clone has at least one wt allele. (I analyzed a lot of clone). Do I have to use a gRNA with a lower ON-Target score ? I cannot use a plasmid because the lipofection efficiency on these cells is 3%. Thank you.
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Hmm, it could be challenging to try and make a less-effective guide RNA. It should be possible but you might spend a lot of time doing so. Adding in a mis-match usually leads to no edits. On that note, could you use your fully-edited cells then add in 1 gene copy that has a mismatch (but still codes for the same protein)?
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I am trying to clone a gRNA (23 bases) into the Crispr cas9 pDirect23A vector by using restriction enzyme cloning. I have performed a complete experiment, starting with plasmid isolation, restriction digestion using AarI enzyme, gel purification of the digested vector, followed by annealing, ligation, and final transformation in the DH5α strain, for three times. Each time I get transformed colonies but could not get the desired sequence of insert in the transformed colonies. There is the insertion of some non-specific bases in between the restriction-digested fragments of the vector rather than gRNA, as confirmed by sequencing. I think I am missing some critical step in the complete procedure of cloning, but I could not find it. Can anyone help me with the procedure?
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I have selected unique restriction site in the plasmid i. e. AarI
I have used fast AP alkaline phosphatase after digestion
I have synthesized a 23bp sequence which contain 20 bp gRNA and the flanking region (3bp) by@@ AarI digestion site
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Hello,
I am trying to clone 1334 bp insert into 6.1 kb vector using HiFi DNA assmebly/Gibson assembly kit. I am taking 100ng of vector and 1:5 molar ratio of vector: insert and 4 hrs of ligation at 50 C. After transformation using NEB DH5 alpha competent cells I am not getting any colonies after overnight incubation. What could be the possible reason? How can i optimize reaction?
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Alexandra Johnson Thanks for your suggestion I will try.
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Hi, may I know
  1. I want to fuse my protein with affinity tag for protein purification in Expi293, how can I determine and visualise how does a N or C terminal tag affect my proteins (structure, stability, folding, expression, is the tag buried and not exposed)? e.g. Alphafold.
  2. Is two types of tag always works better than one type of tag (e.g., His-Strep vs His)
Thank you for any assistance!
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Hey there,
I agree with the previous answers on how to determine how the tag location may influence your protein. Since the effect of the tag very much depends on the properties of the protein, I would suggest making a few predictions with Alphafold like Tim mentioned, but at the end the only way you can be sure on how the tagged protein behaves will be by expressing it and trying it out. If in your Alphafold predictions you see some issues, I suggest putting a linker between your target protein and the tag to check if it improves tag accessibility etc.
For tags, I would recommend using a Strep-tagII or Twin-Strep-tag rather than a His-tag. The Strep-tag usually does not interfere with protein structure and function, and factors like competing with metal cofactors of the proteins are not a concern with Strep-tag. In mammalia cell cultures, proteins native to the host that have histidine residues can often show up as impurities in His-purification, but this is not an issue with Strep-tag purification. So if you would prefer having one rather than two tags, I would always recommend the Strep-tag for a higher purity and yield, especially when working in mammalia, yeast or insect cells.
Good luck!
Christel
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Hi, Does anyone know pet28 cloning vecot can express in E.coli? I have to clone two genes in to pseudomonas and I dont know whether pet28 plasmid derived from E.coli can whould be successful. On the other hand, Pet28 plasmid can replicate in pseudomonas?
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If I remember correctly, vectors with the pBR322/pMB1/ColE1/pUC* origin of replication (different names for the same plasmid oris) don't replicate in Pseudomonas species because a critical RNA for plasmid replication is not expressed. The Pseudomonas DNA-directed RNA polymerase simply does not recognize the sequence upstream of it as a promoter. The vast majority of vectors out there use replication origins derived from E. coli which either function poorly or not at all outside of the Enterobacterales taxonomic order (which includes E. coli).
Most vectors designed to replicate in Pseudomonas utilize unusual origins of replication, derived from natural broad host range plasmids, which function in many organisms distantly related to each other. These plasmid origins conveniently also replicate in E. coli.
*Technically the pUC sequence has a few mutations to the origin that increase its copy number compared to pBR322/pMB1/ColE1 but it's essentially the same otherwise.
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I have two different construct:
1. Insert 1 (~3kb) at XhoI and ApaI site in NCVB vector (9.7 kb).
2. Insert 2 (~1.7kb) at XhoI and ApaI site in in pENTR/D vector (2.6kb)
I want to replace Insert 1 with Insert 2. I have already tried restriction digestion followed by Quick ligase (NEB) or T4 ligase (Thermo). I even tried using CIP as I got a large number of self-colonies without it. However, upon CIP usage, no colonies were seen in either plates (self and test). Kindly suggest ways to go about this cloning. The final product that I want is
“Insert2 in NCVB vector at XhoI and ApaI sites”.
PS: I am using ultra-competent DH5a (CSHL protocol) for all my cloning.
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There was an internal XhoI site in the vector which was creating issue. I got the clone by using different sites and another shuttle vector. Thanks everyone for their valuable suggestions.
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Kirk Aanes must have been kind for Mulan (his ex) to speak well of him. "Just learned of the passing of Kirk Aanes. My condolences to his famiy and loved ones. He was a good soul. RIP, dear one"( https://twitter.com/MingNa/status/431264318701584384?s=19 ).
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Some wanted to reproduce but, never were able.
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To do gibson assembly cloning can I use DH5alpha strain of E. coli. for transformation?
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Yes you can
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I isolated my cloned plasmid from TOP10 cells using invitrogen plasmid miniprep kit and eluted it in TE buffer. But I noticed that contrary to my plasmid aliquot, it does not freeze at -20 degree celsius. I am asking this question because latter when i did confirmatory restriction, i am getting incomplete digestion, and i am linking it with the fact that my cloned plasmid aliquot does not freeze (i am questioning what else is there in it, or is it that i preheated the TE buffer at 70 degree celsius before elution step, so that affected the buffer in someway), thanks in advance for the guidance !!
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I think that you did not dry enough the coloumn after the washing steps and some traces of ethanol or isopropanol (those are commonilly prenset in the washing buffers) remains into your sample.
best
Manuele
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My questions are ...
1. What are sequencing primers ? Are these primers used in DNA sequencing ?
2. why sometimes PCR products are cloned and transformed into competent bacterial cells. What is the necessity of this step ? Why cant we directly sequence the PCR products ?
3. In my earlier works, i did 16s rRNA PCR amplification, Run gel, Excise the band , purification using kit and sent for 2nd party sequencing ?
kindly answer
thank you
regards, Kishor
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1. Sequencing primers are short, single-stranded DNA sequences designed to bind specifically to the target region of DNA to initiate the sequencing reaction. They play a crucial role in DNA sequencing as they provide the starting point for DNA polymerase to begin synthesis. These primers are indeed used in DNA sequencing, particularly in methods such as Sanger sequencing and next-generation sequencing (NGS). Sequencing primers ensure the accurate reading of the DNA sequence by guiding the sequencing machinery to the correct location on the DNA template [1][2].
2. PCR products are sometimes cloned and transformed into competent bacterial cells to create a permanent, stable source of the amplified DNA. This step is necessary for several reasons:
- Stability and Quantity: Cloning allows for the production of large quantities of the DNA of interest. Directly sequencing PCR products might not provide enough material for repeated or extensive analysis.
- Error Reduction: PCR can introduce errors or produce mixed populations of DNA molecules, especially when dealing with complex samples. Cloning into bacteria allows for the isolation of single, uniform DNA molecules, reducing the risk of sequencing errors caused by PCR-induced artifacts [8][9].
- Ease of Manipulation: Cloned DNA can be easily manipulated, sequenced, and stored for long periods, facilitating further studies and applications [8].
Direct sequencing of PCR products is possible but may be less reliable due to the presence of mixed or erroneous sequences, especially in complex or high-throughput settings [3][5].
3. In your earlier works involving 16S rRNA PCR amplification, running a gel, excising the band, purifying it using a kit, and sending it for second-party sequencing is a common and effective workflow. This method helps ensure that the correct amplicon is sequenced by isolating the specific band corresponding to the 16S rRNA gene from the gel. This step is crucial for:
-Purity: Gel excision and purification remove non-specific products and primer dimers, ensuring that the sequenced DNA is the intended target [4].
- Accuracy: By isolating the correct band, you minimize the risk of sequencing errors and improve the accuracy of the microbial community analysis [4][7].
This workflow aligns with the need to obtain high-quality, specific sequences for accurate microbial profiling, as demonstrated in studies comparing different sequencing and analysis methods [1][2][6].
Reference
[1] Tremblay, J., Singh, K., Fern, A., Kirton, E., He, S., Woyke, T., Lee, J., Chen, F., Dangl, J., & Tringe, S. (2015). Primer and platform effects on 16S rRNA tag sequencing. Frontiers in Microbiology, 6.
[2] Schloss, P., Jenior, M. L., Koumpouras, C., Westcott, S. L., & Highlander, S. (2016). Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system. PeerJ, 4.
[3] Gloor, G., Hummelen, R., Macklaim, J. M., Dickson, R. J., Fernandes, A., MacPhee, R. A., & Reid, G. (2010). Microbiome Profiling by Illumina Sequencing of Combinatorial Sequence-Tagged PCR Products. PLoS ONE, 5.
[4] Nübel, U., Garcia-Pichel, F., & Muyzer, G. (1997). PCR primers to amplify 16S rRNA genes from cyanobacteria. Applied and Environmental Microbiology, 63, 3327 - 3332.
[5] O'Donnell, J. L., Kelly, R., Lowell, N., & Port, J. A. (2016). Indexed PCR Primers Induce Template-Specific Bias in Large-Scale DNA Sequencing Studies. PLoS ONE, 11.
[6] Xue, Z., Kable, M. E., & Marco, M. (2018). Impact of DNA Sequencing and Analysis Methods on 16S rRNA Gene Bacterial Community Analysis of Dairy Products. mSphere, 3.
[7] McGovern, E., Waters, S., Blackshields, G., & McCabe, M. (2018). Evaluating Established Methods for Rumen 16S rRNA Amplicon Sequencing With Mock Microbial Populations. Frontiers in Microbiology, 9.
[8] Finney, M., Nisson, P., & Rashtchian, A. (2001). Molecular cloning of PCR products.. Current protocols in molecular biology, Chapter 15, Unit 15.4 .
[9] Costa, G. L., & Weiner, M. (2006). Bidirectional cloning of PCR products.. CSH protocols, 2006 1.
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I did whole plasmid sequencing of a plasmid I received from my colleagues when the result came out why was everything reversed in it? Meaning the format should be 5'promoter-gene-terminator- t7-3'. but instead, it came as t7-terminator-gene-promoter. I need to clone my gene between the t7 and the terminator. How should I clone it? Should I do everything reversed as I have to keep the format as 5'-gene-3'.
Please suggest.
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Sequence assembly software usually assigns the + and - strands arbitrarily and sometimes this is different from how you would personally assign it, since the software doesn't really know what it's "supposed" to look like. If you take it into plasmid annotation software and flip the +/- strands does that fix the issue?
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Hi,
My question will be at a very fundamental level about heterologous gene expression,
For any (heterologous) gene we insert between any promoter and the corresponding terminator sequence, is it possible to express it successfully at low or high levels in a host cell?
For heterologous expression, should I either clone any gene with its natural promoter and terminator sequences or insert it into the 'coding region' of a P+coding region+T sequence already existing/working in the host cell's genome?
Many thanks,
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Abdurrahman Aygül It means, Targeted knock-in vs Stable-transgenic expression.
Targeted knock-in - Difficult to perform, can use native elements, less prone to genomic silencing, mostly weaker expression than the TG.
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Asper discussion, First time my clonning was failed becuase the first 45 nucleotide was not cloned in pet30 a vector then I choosed the different clone of 1:1 ration fraction, I got the size of purified protein near to the expected size 17.7kda and very thick band but there was also thin nonspecfic protein band saw in Western blot result whose size is more than 20kda but less than 23 kda but size of band is very thin. can anyone please tell me ? what could be the reason ?
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Heartiest thanks Dr, J.Stolz for your such great response
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My vector size 5.1kb and insert size is 4kb. I performed double digestion of vector and insert, ran them on the gel and then extracted them from the gel. 260/280 ratio was good. Then I did ligation using T4 ligation at 4C and transformed into E. coli DH5a. After 24hr, I did not see any colony. I left the plates for another 2 more days in the incubator. Now I see some tiny colonies. My question is -
1) Is there any way to check whether ligation or transformation is the problem?
2) The OD600 of competent cells was about 0.9. Would that be an issue?
3)Are the tiny colonies after 3 days transformed colonies or not?
4) Any suggestion for successful cloning with my vector and insert sizes?
Thanks in advance
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Abdullah Al Tarique for heat shock, the cells are sensitive to OD600, especially if you grow them at 37°C (see Fig. 3 in this paper ). So next time, grow them at lower temperature and check the OD600 carefully.
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Hi all, I have been facing problems with colony pcr for agrobacterium colonies. I did site directed mutagenesis, cloned to pcambia, sequenced and confirmed and then transformed to Agrobacterium. Then I got many colonies in selective media (Rif, Gent, Kan) but couldn't confirm which colony has the right plasmid with insert. For this, I tried colony PCR. The positive control (plasmid that I transformed) shows band but for colonies, no band. I took the colony in 10 microlitre water and then boiled for 10 mins and then added 3 microlitre from that as template. Could anyone suggest what to do? I know miniprep plasmid isolation would not be easy for Agrobacterium. Without confirming also, I tried to proceed with one colony but then could not express in Nicotiana.
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Here is what I do in E.coli not in Agrobacterium,I hope this helps you:
Put little colony in 10 ul water and then added 1.5 microlitre from that as template.If getting band with correct length, then sent to company for sequencing.
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I am using ImmunoCult™ Human CD3/CD28 T Cell Activator (Semcell, Catalog #10971) to activate PBMC and expand T cell. but the T cell activator contains an anti-CD3 antibody clone that fully or partially blocks all anti-CD3 antibody clones used to assess CD3 expression by flow cytometry.
Are there any other ways to avoid the interference of CD3 in the CD3/CD28 T Cell Activator it I have to gate CD3 by FACS? Is it possible to avoid this if I use a different clone No. of CD3 for FACS analysis?
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Jessica M. Reel Thanks a lot! It appears this cannot be avoided, I'll try CD4 and CD8 or others instead.
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I have a protein that is 117 amino acids long, with a 21 amino acid transmembrane domain at the C-terminus. I've cloned this protein into the pET28 expression vector in BL21, but am not seeing any protein expression. Do you think the transmembrane domain could be the reason for the lack of expression? What strategies can I try to improve the expression of this transmembrane protein?
## Background
- Protein length: 117 amino acids (from virus)
- Transmembrane domain : C-terminus, length: 21 amino acids
- Expression vector used: pET28
host:BL21
- Current expression status: No detectable protein expression
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Hello, I agree with Manuele Martinelli's opinion. Most membrane proteins have pretty low expression in E. coli. When I purified membrane proteins in E. coli, I used the inclusion body fraction and membrane fraction to purify my protein. After purifying the fractions using Ni-NTA, I finally saw my protein's thin band on the SDS-PAGE gel. You might not see your protein band in the supernatant after cell lysis. But, you might see your protein band after purification if using the inclusion body fraction and membrane fraction.
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My vector size is 5.1kb and insert size is 4kb. Initially, I performed digestion of the insert from a different plasmid and the vector using KpnI-NotI. I checked them on the gel, excise and extract the band from the gel. I aimed ligation 1:3 and 1:5 at 4C and 16C overnight and RT 1hr ligation. I used fresh enzyme and buffer. I performed transformation in Stellar cells. However, I got very few transformed colonies on plates where ligation was performed 1:3 at 16C ON. Recovery of Stellar cells was great. Unfortunately, the transformed colonies do not contain the insert. They carried the empty vectors. Any thoughts?
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Well, the quick answer is the insert didn't get successfully ligated in, as you already know. Ok, now for some thoughts as to what might be going on and what could help.
Your insert and vector are nearly the same size, might be worth trying a 1:1 molar ratio. Try for about 50 ng of vector per 3 Kb of backbone size per ligation.
Did you run any ligation controls? If you want to know how much background (uncut or cut then self-ligated) you can add in two reactions. First one is cut vector (no insert) with ligase and ligase buffer, that lets you know how much vector was uncut or stuck back to itself. Second one is cut vector (no insert) and no ligase but with ligase buffer (just to keep conditions the same), that lets you know how much vector was uncut. Transform these control reactions along with your actual ligation. Ideally, you get 0 to few colonies on the control plates, and more on the real ligation plate. In reality, there are usually some colonies on all the plates.
Do you use two different restriction enzymes for the two insert ends? Are both sticky? It can be a challenge if one or more leaves blunt ends.
Another issue can be if your donor plasmid and destination plasmid have the same selection marker. I had this happen before and I would get back the original insert vector. I added in a third enzyme to cut in the vector backbone (but not in the insert). That did work.
Hope this all helps!
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Some journals seems to be fake..cloned
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Cloned is not the right word since this would imply that there is a legit version of this journal “International Journal of Scientific Research in Engineering and Management”. I am afraid that this is an example of a predatory journal. There are numerous red flags:
-First of all, the prominently mentioned UGC-CARE is false. They are not listed as can be checked here https://ugccare.unipune.ac.in/apps1/home/index
-The prominently mentioned impact factor (https://ijsrem.com ) is at least misleading since they are not indexed in one of Clarivate’s indexes which can be checked here https://mjl.clarivate.com/home
-Even stronger the SJIF factor is a notorious example of a so-called misleading metric (https://beallslist.net/misleading-metrics/ ) often used by predatory journals/publishers
-The same is true for I2OR and CiteFactor
-Disclaimar [sic] on the home page https://ijsrem.com and Copyright Infra