- Hao Zhang added an answer:2Does Uracil-DNA Glycosylase (UDG) block the function of Q5 DNA polymerase?
after 30min of UGD disposal, I took 15 uL and 10 uL from the previous 50uL reaction, and used Q5 (NEB) for the next step PCR, yet with no product at all.
THX! It really helps! I myself also just found out I have asked a silly question...It is reported that pfu, Klenow Taq and Vent are compatible, which neither do we have QAQ, so I suppose Taq enzyme should operate similarly. I checked the lab stock and found out LA Taq (RR02MA) might be a good candidate.... But the protocol on their website is kind of a chaos, I can't find code RR02MA product. I'm still searching. And if it doesn't work I'll consider buying pfu on Monday.Following
- Anirudh Gautam M added an answer:8Is there any way to avoid cleavage of eukaryote protein expressed in E coli?
Hello, I am trying to express eukaryote protein (36 KDa) in E coli ( BL21 and Rosetta) by using different vectors (pGEX-4T-2 and pET-28a) and tags (GST and His- tag, respectively). However, more than 70% of my protein was cleavaged from the middle! Has somebody faced same issue? Is there any approach to stop cutting my protein? Thanks in advance for any help.
If your protein is being cleaved inside the bacteria, then how many bands of protein are you getting on your PAGE gel ? If after a mass spec, you can identify where your protein is being cleaved inside the bacteria, you could try to identify the cleavage site that is present in your protein and change a residue to its nearest amino acid with similar properties (with a similar functional group like -SH with -OH etc) . Now with this residues change the bacterial cell should no longer be able to cleave your protein and the overall structure of the protein does not get largely affected.
I hope this helps . If the its to be mutated is of importance, I am not very sure what can be done. Have you codon optimised your expressing sequence to bacterial codons ?Following
- Apurva Agrawal added an answer:8Can sarkosyl be used for the solubilization of inclusion Bodies?
Dear Esteemed Researchers,
I am trying to solubilize a membrane protein that forms inclusion body when expressed heterologously in E. coli cells. As such, I tried various detergents to screen the solubility of my protein. Intriguingly, I found that my protein was able to be solubilized in the presence of both ionic (>0.1%Sarkosyl) and non-ionic detergents (1% Octyl-beta Glucoside and DDM). However, because the price of the non-ionic detergents is horrendously expensive, therefore, if possible, I wish to use this non-ionic detergent in my crystallization trials and employ other detergents such as Sarkosyl in combination with TritonX-100 and CHAPS for extraction and purification purposes.
Now, what disturbs me here is the use of Sarkosyl. I have read articles that the use of high concentrations of anionic detergent such as SDS and Sarkosyl may denature and may distort the function and structure of proteins. In one of the paper that I read published in Biotechniques (attached herewith this question thread), the authors have used up to 10% Sarkosyl to solubilize inclusion bodies of various poly-histidine tagged and MBP tagged proteins and subsequently diluted the sarkosyl to 1% to purify the protein in the presence of Triton X-100 and CHAPS.
So I am confused now whether the use of Sarkosyl at such high concentration will disrupt the native structure of my protein? Since it is a structural protein, therefore, I do not have any assay to validate the biological activity of my protein.
I would highly appreciate your immense insight in providing me with some directions on the use of Sarkosyl in my lysis buffer. Also, in your opinion, what is the best way to measure if my protein is intact despite the use Sarkosyl?
I would like to add my query as well here in this forum.
One of the proteins that I am looking to study is intrinsically in aggregated form in the cell even in normal conditions. Hence, getting a sds- page and western blot for the protein is tough. The cells are animal tissue culture cells.
Can some one guide me with detailed protocol as how to make this protein soluble to be noticed on the western blots.
Thanks in anticipation.
- Thibault G Sana added an answer:10Can anybody give some suggestions for knocking out essential genes in Pseudomonas aeruginosa?
I need to construct a conditional mutant for an essential gene in PA14. At first, I knocked in a copy under pBAD promoter into chromosome via attB site using integration plasmid. Gene expression was confirmed by WB. So, I go ahead to transfer knock-out plasmid by electroporation. This plasmid contains upstream, Gm cassette, and downstream homologous arms. I can get single crossover mutant, which is confirmed by colony PCR with upstream screening primer and Gm internal primer. This PCR product was confirmed by sequencing. This strain was GmrCBCr.
I am very happy to continue to use 10% sucrose+0.2% arabinose to screen double crossover mutant. I got many GmrCBCs colonies. Unfortunately, the gene seemed to restore to wild type. Colony PCR using screening primers showed the same size as wild type, which should be 500 bp shorter.
I am confused. How can a single crossover go back to wild type, but still with Gm resistance? I repeated several times. The same thing happened. what should I do? can anybody give me some suggestions? I appreciate your nice help.
Congratulations! Happy to see that I could help.
- James E Lincoln added an answer:3What is the best linker for recombinant protein expression in a plant platform?
When I use linker with long sequence (288bp) between protein and 6H tag , the minimum free energy of folding related to mRNA was decreased by mfold software which indicat stability of secondary structure of mRNA. But I would to know this long linker will influence protein expression and has sterical interference for protein folding or not.
I am guessing that a minor addition to one end of the protein will not affect the abundance of the protein.Following
- Daniel Lorenz Winter added an answer:7Do We have any primer (Oligos) repository for full length cloning of genes?
Could anyone tell me any primers repository for full-length cloning of genes, like we have for real-time primers?
Sorry I haven't replied in such a long time, and I believe by now you might have solved your issue. But depending on what you want the variants for, you could simply clone the entire genes and use the appropriate mutagenesis techniques to recreate these variants (or more simply, order the entire cDNA).Following
- Pedro Luis Ramos-Gonzalez added an answer:7How can I clone 9.7kb single stranded positive rna of potyvirus ?
i have designed degenerate primer and amplified product (700 bp) is cloned and sequenced. but now how can i proceed for for cloning of rest (9.0kb) of the the genome.....
I recommend reading this
Nagata T, Inoue-Nagata AK. Simplified methods for the construction of RNA and
DNA virus infectious clones. Methods Mol Biol. 2015;1236:241-54. doi:
10.1007/978-1-4939-1743-3_18. PubMed PMID: 25287508.Following
- Aditi Singh added an answer:9Why do we see non-specific colonies on drop out medium plates in DNA containing sample and No DNA control.?
Why do we get non-specific colonies on SD-Ura and similar dropout medium plates in DNA containing and No DNA samples after the yeast transformation (LiAc-PEG method)? If transformation is successful then one should see it under the microscope (GFP for example) but these non-specific colonies do not show that signal however they can grow happily under the selection pressure (URA containing medium e.g.).When I check non-specific colonies of the yeast (S.cerevisiae) under the microscope they show presence of some kind of structures inside the cell. Does anybody know what those structures are? How to avoid this non-specific colonies?Following
- Nagabushan Reddy added an answer:17Why do I keep getting multiple bands for my plasmid extraction even after using QIAGEN miniprep?My vector is pT7 FLAG2 sized 4.8kbp and my insert is roughly 2-3kbp. I grew them in BL21 and sub-cultured them overnight with ampicilin before extracting them. After that I digested them with my enzymes (Bglii and Hindiii) from ThermoScientific.
My hunch is that I have supercoils and nicked plasmids but the bands I have don't correspond to the size of my plasmid and insert (which are around 7kbp).
I also tried doing overnight digestion and still have the same result. Any idea how I can eliminate the extra bands and whats going on? Your help is highly, greatly appreciated.
Hi , Is your markers the same in both the gels? Because your bands are of different sizes.Following
- Jeza M Abdul aziz added an answer:18Contaminants in DNA?Low 260/280 in DNA isolated from liver tissue with Qiagen DNeasy. I am isolating total DNA from liver tissue for downstream use in exomic sequencing. I need a final quantity of 3.6 micrograms at a concentration of 60ng/ul. My yield looks high but my nanodrop OD looks bad. With other samples i have gotten 260/280 of 1.9-2 but now its lower, at 1.6. The minimum ratio needed for this application is 1.80. I think this is protein contamination, though I am not sure. The 260/230 is also <1, which I've read may be due to contamination with salts. I added RNAse to this purification, though it didn't seem any different from the identical one without RNAse. Should I perform the elution again? Should I add more proteinase K? Digest for a longer period of time? I have plenty of yield so I could do part/all of it again, but don't want to waste a lot of spin columns. I also thought that perhaps my elution was too concentrated as I used only 100uL instead of the suggested 200uL to elute so I added another 100uL of the AE (elution buffer) to my final sample to see if that helped and it didn't seem to.
Secondary measure of nucleic acid purity is 260/230: ratio of samples absorbance at 260 and 230 nm. The 260/230 values for “pure” nucleic acid are often higher than the respective 260/280 values and they are commonly in the range of 1.8-2.2, If the ratio is appreciably lower, this may indicate the presence of co-purified contaminants.Following
- Steven Larsen added an answer:14Is there anything wrong in the coloning methods for protein overexpression?
I used NcoI and XhoI for the recombinant protein construction in pET28, and I found it had no any frame shift when simulate construction map in SnapGene software.
The problem is I always cannot get overexpessed protein after IPTG induction, not just one protien but all of them.
In case there is any issue about codon usage bias (my target gene is not from E. coli, my expression host is BL21, and the induction concentration of IPTG was from 0.5mM to 1mM), the prediction of relative codon bias scores had no significance difference.
And I pretty sure my targets are not toxic proteins.
So, Does anyone have experience on this problem, thank you.
Are you sure you are not getting any expression? It could be that the proteins are insoluble, you can check by western blot of the whole cell extract after lysis in SDS-sample buffer. If that is the problem then the suggestions by Roger bradley Dodd may help.
Also you only said you checked for frame shift with genesnap, if you didn't sequence the plasmid you need to.Following
- Florian Nadler added an answer:15How can I clone 2 separate PCR fragments into a new vector?
Hello all. I am new to the cloning world and would like to clone 2 different PCR products into a new vecor. I have designed PCR primers to add an EcoRI restriction cut site followed by my first gene of interest followed by a BamHI restriction cut site (for a total of ~1kb long for PCr product 1). That the next gene of interest will be cloned using primers to add a BamHI cut site followed by my gene of interest and then a XhoI cut site (total of ~2kb for PCR fragment 2). I want to ligate these 2 fragments together at the BamHI sticcky ends and then insert them into a new pCS2+ vector (which I will cut at the EcoRI and XhoI cut sites). My questions are: 1) After I PCR the two fragments separately (using their respective DNA constructs as a template) do I do a restriction enzyme digest and run on an agorose gel to purify the fragments with new sticky ends? 2) Should I ligate the 2 new fragments together first and then insert into the new pCS2+ vector (digested with EcoRI and XhoI) or can I ligate all 3 together at once?
There is also a technology called SLiCE (Seamless Ligation Cloning Extract), which does essentially the same as Gibson Assembly, only hundreds of times cheaper. You just add homologous regions to your primers, as you would do for Gibson Assenbly, and then treat all of your fragments with an E. coli-extract (CelLysis B works best for the extract generation). It´s one hour at 37°C and works for up to 4 fragments simultaneously.Following
- Mohammad Ayoub added an answer:10Can anyone help me with DpnI-mediated site directed mutagenesis?
I am trying to generate cell lines carrying phosphomimetic substitutions. I have 5 primer pairs, each are ~60 bases.
I am using 2.5 micro Molar (uM) forward and the same for rev. primer amd 50ng template DNA. Also I have added 3% DMSO .
The PCR cycle I am following is
Initial denaturation at 98C for 1min
98C for 25 sec
72-70-68C for 25s
72C for 5m
final extension at 72 for 10m.
DpnI digestion for 2hrs at 37C.
I used competent E. coli. I have seen colonies (But also I have seen background in -ve ctrl plate!)
After mini-prepping and seq, I found I don't have any correct insertion. All of them were exactly like WT sequence!!
What do you think I should modify in this protocol to get correct insertions?
I would appreciate your add!
I am so sorry for not following your respectful comments.
I got all the mutants now!!
I have done two things which are the causes of previous failed mutagenesis.
1- I have used very very high annealing temperature, for PCR-based mutagenesis using long primers (~60 bp), I should use maximum 55C.
2- Pfu polymerase works perfectly, unlike Phusion polymerase, with blunt-end primers.
Thank you everybody for your suggestions!Following
- Jiexi Hou added an answer:5Does anyone know what will happen if there are two promoters in one construct?
pMDC85 promoter contains 2x35s constitutive promoter and I've put in another tissue-specific promoter down-stream of 35s. Now I am thinking what tissues in plants will GFP label?
Thanks Yuan-Yeu. I will let you know if I can get any good ideas after I read this paper.Following
- Leong Chia Choong added an answer:14Can anybody help with my ligation as it has failed multiple times(No colonies) ?
My goi is base on PCR cloning, as my primer design has 2 restriction enzyme site, which amplify my product with the sites (EcoRI and SapI). After digesting both my PCR product and plasmid, I ligated both of it with 3:1 vector to insert formula, and even tried with 6:1 but it is no working. My gene of interest is Kanamycin and my vector has ampicilin, so when doing selection on agar plate, the agar contain both the antibiotics.
Thank you for answering my question!
After multiple trial and error I have successfully ligated my product, and my previous failure might be due to that I have skipped a single step which is heat deactivation of my enzyme before transforming into competent E.coli cell.Following
- Charitha Gangadharan added an answer:7Can we use a lentiviral (promoter/ expression) construct for transfection in MCF7 cells by lipofectamine based method?
Can we use a lentiviral (promoter/ expression) construct for transfection in MCF7 cells by lipofectamine based method? If not how can I modify the construct for an antibiotic based selection construct? I plan to do a transient transfection with a GFP/LUC construct. The gene of my interest is already cloned in lentiviral construct. How can I subclone to a PEGFP/ promoter LUC construct?
Thank you all for the valuble inputs given and the time you took to answer my questionFollowing
- Sharmila Narayanan added an answer:7Can you help me with transient transfection?
We have cloned our gene in mammalian expression vector pEGFN1 vector by introducing a stop codon between the gene of interest and EGFP sequence so that no GFP is expressed. While performing transient transfection in HeLa and A-549, we used pEGFPN1 vector as control (Where GFP is expressed). It has been observed that the vector is also showing toxicity but slightly lesser than the gene of interest containing pEGFPN1. This we got to know by doing MTT after 48 hours of incubation. Does anyone have any idea, how can I solve this problem? Or what is going wrong that can be corrected.
@Paritosh . We isolate plasmid using kit (Sigma) and for transfection we use Serum free media onlyFollowing
- Andrew Jenkins added an answer:9Are you familiar with restriction digest with a single enzyme?
I am cloning out a fragment, using SacII, containing the ORF of my interest and cloning it into a SacII site in the multiple cloning site of my vector. The insert can obviously orientate itself in one of to ways in this case but I need it to be in the "correct" orientation. There are no other unique enzyme pairs flanking the ORF or in the MCS. Is there a way that I can encourage ligation of the "correct" product? Thank you.
If you're only getting the 'wrong' orientation - presumably meaning that the promoter transcribes the antisense strand - then it suggests that your product is toxic when overexpressed. You will need to keep the gene 'turned off' until it is time to induce it.
If for example, you are cloning in a plasmid of the pUC family with the gene under control of the lac operon, keep the lac operon turned off by adding glucose and leaving out IPTG. The drawback with this is that you lose the blue-white screening, but if you're using dephosphorylated vector this shouldn't be too much of a problem.
Another trick you might like to try is to deliberately pick small colonies. This will work if your product is mildly toxic and reduces growth rate without actually killing the cells.Following
- Nicolas Grandchamp added an answer:11Apart from HEK 293T cells, can HEK 293 also produce lentiviral vectors?
I use CaPO4 precipitation transfect 293T to produce lentiviral vectors(packing vectors are pMDL, pRev and pVSVG). However, I take the 293 other than 293T. So I am worry about whether 293 can produce the virus I need. Any suggestion will appreciated!
Agree with Aron, you can produce efficient LV with 293 but it is with 293T you will produce the most efficient LV.Following
- Mark K Chee added an answer:14What are the strategies for creation of constitutively active mutants of a protein?I am working on a protein which becomes active when phosphorylated at a particular tyrosine site. I am looking for methods/ strategies as how to make constitutively active mutant for this protein. Please suggest some.
An unnatural amino acid that mimics phosphotyrosine
- Rajendra Kumar Chauhan added an answer:7How can I check the large DNA sequence (in an expression vector) ?
I want to check gene sequence that is a large size (3.7kb). This gene is located in MCS of pQE9 vector (Qiagen).
How many primers that i should design for check full gene ?
Do i need to sequence double strand (using both forward primer and reverse primer)
Thank you for your suggestion
There are different programmes for sequencing a little bit longer stretches of sequences,We generally use Rapid for sequencing upto 700bp.The sequencing run takes a little longer than usual,but you get longer reads.So as other people have pointed to use 4-5 primer sets would generally solve your sequencing dillema.
Combination of primers from the vector and within your insert would be the best.Following
- Ravinder Kaundal added an answer:8Could anyone recommend a stable high transfection efficiency mouse cell line that I could use?I've been trying to transfect MEF cells using several methods and I did not get a very high efficiency (we have the Nucleofection kit too but there are many constructs that I need to transfect and it would be too expensive ) therefore I would like to try a different mouse cell line that shows a higher transfection efficiency. Could anyone suggest any?
I would truly appreciate your help. Thank you.
What was the transaction efficiency of MEF cell line with lipfectamine 2000?Following
- Bhanu P Jagilinki added an answer:15Is there any bacterial strain that can maximize the expression of recombinant protein?
We are trying to express a fusion of His tag protein in E. coli BL21 DE3 and Rosetta DE3 strains. Our fusion protein (~23 KDa in pET28a) does not appear.
The gene sequence is in frame with the His tag and we try change temperature and different concentrations of IPTG to induce the protein. Somebody have any suggestion for this problem?
Did u checked, if ur protein is going into inclusion bodies?? After sonication, centrifuge @15k rpm and load both pellet and supernatant separately.
For pET-28 constructs, prefer Rosetta-2DE3 where u have to add both Kanamycin and Chloramphenicol.Following
- Paola Campomenosi added an answer:16Is my gene amplifying?
I have a gene(wild type) cloned in pBAD18 Kanamycin resistant plasmid. The gene is 2185 bp. And the plasmid is 5437 bp. I wanted to insert a 6X His tag at the C-terminal end of the protein of this gene. I found that the last codon codes itself for a His. So I followed the general method for designing a primer i.e.: Last codon(CAT)-6X His codon (CAT or CAC)- Stop codon-XbaI recognition sequence. The primer length is 30 bp with a Tm of 62 degrees approx and the forward primer I used was 28 bp and has a EcoRI recognition site with Tm 59 degrees approx.
I have tried amplifying my gene using a gradient PCR vaying the annealing temperature (see gel image). I expect to get the product in between 2Kb and 3Kb(2203 bp). But I have a range of variation in amplification above 10 Kb. What is getting amplified is my question? Is my gene at all getting amplified? I dont think so. What is going wrong.
I am using Pfu polymerase with 30 steps amplification. I have done gradient PCR with variation in annealing please see gel image attached. PCR program:
Initial denaturation: 95 degree- 3 min
denaturation: 95 degree- 30 sec
annealing: gradient- 55- 64 degree- 30 sec
extension: 72 degree- 3 min 30 sec
Final extension: 8 min
Store: 4 degree.
Notice the amplified product that I am getting is above 10 Kb.- DNA Ladder used is 1Kb NEB ladder.
please suggest ways I can get my gene amplified for cloning. I want my insert to have a 5'-EcoRI and 3'-6X His-XbaI.
1) how much of your starting plasmid template do you use for PCR? Have you tried to load it on the agarose gel to check that the bands you observe are not the original template?
2) it seems to me that 30 nt are too short for your primer: it must contain 6 nt for the restriction site, 3 for the stop codon, 5(or 6) x3 His codons = 15-18 and finally the sequence complementary to your target... try to extend the complementary part at least to 18-20 nt.
(and beware you must not indicate a primer in bp, as it is single stranded! ;-))
- Daniel M Cohen added an answer:9Does pIRES2 express more EGFP than the mRNA before the IRES?
I've subcloned all of my mRNAs into pIRES2 (CMV:mRNA-IRES-EGFP) for expression in cell culture (CHO and TSA cells) and electrophysiology. I have been getting a lot of bright green cells with absolutely no measurable expression of my mRNA: about 50% of the green cells show the expected current, the other 50% react like an untransfected cell.
Is this a common problem with the pIRES?
The IRES-GFP cassette is designed to work in conjunction with a stop codon in your GOI. If this is absent, your GOI may not express properly. Based on what you describe, it sounds like the co-transfection experiment was a better setup. Perhaps the transfection efficiency of the IRES2-GFP plasmid is simply less good in your hands either due to increased plasmid size or due to variability in the quality of the purified DNA.Following
- Indrajit Sahu added an answer:3Can I convert the validated efficient shRNA sequence of one specific gene to siRNA sequence? If yes, how can I achieve it?
I have the validated efficient shRNA sequence targeted for one specific gene and its length is 21nt ,which is suitable for lentivirus knockdown pLKO.1 vector.
But now I want to knockdown this gene by siRNA or stable shRNA transfection by electroporation with pSUPER vector, for which I need the efficient 19nt sequence.
Can I convert the validated efficient shRNA sequence of one specific gene to siRNA sequence? If yes, how to achieve it?
YES, of course.
As per my knowledge is concerned if your 21-shRNA has efficiently worked, then you can directly convert into a 21 long siRNA. There is no need of modification at all. After all your shDNA-pLKO construct eventually converted into a single standard 21 base long siRNA, which had given u good results. And of course u just need to change the T to U.
Importantly, u need to keep the "2 base" (most preferably AT/AU) 3' overhang after the 19base antisense sequence. That will increase the activity (final degradation of mRNA by RISK-complex).
I have tried it in my lab and it works better.Following
- Touraj Farazmandfar added an answer:5What are the precise sequences for Age1 site to add to oligos for shRNA cloning in pLKO.1?
I'm planning to do shRNA cloning in the pLKO.1 for lentivirus production. i read the protocol provided by addgene et some others. There is a little that I don't understand regarding the Age1 restriction site. The protocol is based on annealed oligos that you put the open vector. The bases recommended to add at the 5' side of the forward oligo are CCGG. but the restriction site normally is CCGGT. Why we don't put the "T" at the end of this site whereas they put a full site for EcoR1? Hope i was clear . thanks in advance
order synthetic oligo cloned in your plasmidFollowing
- Nirav Rajivkumar Shah added an answer:1Is pSuper.neo.gfp vector a suitable system for stable microRNA over-expression or knockdown for its functional characterization?
I am planning to use pSuper vector system for over-expressing and knocking down a microRNA in breast cancer cells. Although it was initially designed for the purpose of siRNA expression, I am not quite sure about its suitability as far as miRNA expression is concerned. Also, I also would like to know that whether cloning a sequence antisense to a particular miRNA in the same system can efficiently result in knockdown of that miRNA.
I work with miRNA and to over-express, a particular miRNA first I amplified ~350 bp region containing pre-miRNA with my desired restriction site on both ends. I cloned this into pGEMT-Easy vector and then I cloned that piece into the PCDNA 3.1 + vector (zeocin). I transfected HCC-1954 cells with this cloned vector (0.5 ugs) and collected around 25 individual clones. It is extremely important to collect more clones since expression of your miRNA will vary from clone to clone. Based on Real time data, I have approximately 800-4000 fold up regulation of my miRNA of interest. For knock down the most suitable approach will be to use sponge. see this paper http://www.jbc.org/content/early/2013/08/06/jbc.M113.491803.full.pdf.
I hope this helps,
- Elena Azhayeva added an answer:2Has anyone used Trimer Codon Oligos and can offer advice?We want to create a semi-random expression library (around 30aa). This could of course be done by using poly-N oligonucleotides, but there is a newer technology where the oligo is synthesized using trimeric nucleotides (codons). This has advantages since you then can choose not to introduce stop-codons, avoid certain amino acids and make customized mixes of codons for certain positions in the oligo.
My question is has anyone experience with these? How long oligo did you order? Any company to recommend?
Metkinen Chemistry provide synthesis of the Trimer phosphoramidites as well as Randomized Oligonucleotide Libraries using them. The length of the oligonucleotide up to 100 b, but number of randomizations is limited to 10-14. You can get more information at www.metkinenchemistry.comFollowing
About Molecular Cloning
A platform to ask questions and discuss the procedures involved in Molecular Cloning.