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Cloning - Science topic
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Questions related to Cloning
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!

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.

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.
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?
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?
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?
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.
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?
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
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?
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.
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
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?
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.
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.
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
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?
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?
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
Does anyone have experience with the Anza™ Restriction Enzyme Cloning System? Have you ever had any problems?
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?
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.
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?
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?
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?
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.
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?
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!!!
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.
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)
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?
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.
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.
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.
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?
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?
Hello, I have a question, I am starting with GWAS analysis.
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?
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.
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Thank you,
Chris

I am going back to recovering some BAC clones for gene targetting. But I cannot find the ordering source. Anyone can help ?
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.
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,
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 :)
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
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.
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?
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.
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).


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.
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.
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.
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.
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.
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.
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?
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?
Hi, may I know
- 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.
- Is two types of tag always works better than one type of tag (e.g., His-Strep vs His)
Thank you for any assistance!
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?
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.
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 ).
Adding to my case: https://www.researchgate.net/publication/381483753_Honor_Kirk_Aanes

To do gibson assembly cloning can I use DH5alpha strain of E. coli. for transformation?
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 !!
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
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.
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,
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 ?
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
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.
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?
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
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?