Dear all,
Recently, we have trouble inserting DNA fragment into vectors by traditional PCR cloning. Please provide any comments and/or suggestions. High appreciate it!
Basically, we want to insert seven different promoter regions (those seven promoter regions are for seven quorum sensing genes) into plasmid, which has fluorescent genes, for our gene expression study. We designed the primer pairs for those seven promoter regions and performed PCR reactions to get the DNA fragments. We cut those DNA fragments (or, the promoter regions) by double enzyme digestion. Our plasmid DNA was also cut by the same restriction enzyme couple. We then performed T4 ligation and sent the recombinant DNA or plasmid to E. coli TOP10 cells via chemotransformation. Four of the seven transformations were successful while the remaining three did not work: From the colonies just after the transformation, we performed colony PCR and got good result. When I subcultured the colonies into new plates, I got many single colonies. I performed colony PCR using those new colonies but did not get any band in the gel. Also, I can isolate plasmid DNA from both the original colonies (the colonies just after the transformation) and the new colonies. However, when I used the isolated plasmid DNA as DNA templates in my PCR reactions, I still get no band in the gel. I have repeated this experiment (double enzyme digestion, ligation, transformation, colony PCR, and/or plasmid DNA PCR) for several times and still have no lucky. We are really confused as for the four successful transformations, we got good results using the same reagents and the same procedures.
For the four successful transformations, the inserts are relatively short (< 300 bp). The three promoter regions that need to be inserted into this vector have the length of 523 bp, 756 bp, and 879 bp. So, I am guessing that the sizes of the DNA fragments/inserts are an issue but I have no clue how to deal with it. I read the comments from the following discussions and followed most of the suggestions:
https://www.neb.com/tools-and-resources/usage-guidelines/tips-for-maximizing-ligation-efficiencies
http://www.protocol-online.org/biology-forums/posts/16853.html
Still, I did not get the desired results.
Thanks!
Hi Chiqian,
I would start in the steps previous to the transformation. Have you checked if the double digestions worked?? Does the plasmid after the double digestion religate (you can perform a test or a control only with the digested plasmid, in case the double digestion worked you shouldn't obtain colonies)? Also you can have problems with the digestion of the PCR fragments. Did you add enough nucleotides at the end of the primer next to the RE recognition sites for the enzymes to cut properly??
You could also optimize insert:vector molar ratios (maybe you did already). A ratio 3:1 is a good starting point.
ng of insert = ((ng of vector * kb size of insert) / kb size of vector)* insert:vector molar ratio
Hope this helps,
Tamara
Digesting PCR product sometimes cause problem because its possible that one of the restriction sites works but others not and in this case you won't be able to see the problem. Therefore, its better to first clone the insert into a cloning vector using one of cloning kits and perform D.digest using the cloning vector as a template. If you get the correct size from the D.digest, it means that there is nothing wrong with your insert (Apart from mutation which is possible to happen if you don't use a proper polymerase but it can also be easily detected by sequencing the insert in the cloning vector). When you are sure about the insert then you can subclone it into the expression vector.
Hope it helps.
Hi Chiqian,
Tamara already raised some important questions. From my experience digestion of the PCR products with 2 enzymes can be very critical. You have to consider that the molarity of target sites is much higher than in the control DNA used do define the units/µl. If your multiple cloning site has a site for a blunt end cutter you reduce the risk (and cost) if you combine such a site with a staggered site. Alternative you may consider Gibson cloning to avoid the digestion step of the PCR products. But as Tamara already mentioned, the double-digest of the vector should be very efficient with both methods..
Personally, I would prefer to prepare small DNA preps after the transformation and characterize them by restriction digest.
I guess you have checked that there are no internal sites for your restriction enzymes in the larger PCR products.
Dear Tamara,
Thanks for the comments. Actually, I have tried different insert: vector molar ratios, including 3:1. I also added enough nucleotides at the end of the primer next to the restriction enzyme recognition sites.
One thing that I am not sure is "Does the plasmid after the double digestion religate". There is no suicide gene in my vector/plasmid, and if the plasmid religate, I think I would still get colonies.
Any comment for that? Thanks a lot!
Dear Soran,
So, your suggestion is that, I might need to clone the insert to a cloning vector (e.g., the one from the TOPT TA Cloning Kit) and them cut it off and subclone it to my target vector. I think that is a good idea and I would like to try that.
Dear Uwe,
Thanks a lot for that. There is no internal cutting site. So, I am very confused by the results. Actually, I always got good colony PCR results after the transformation. However, when I subcultured the colonies that yielded good results in colony PCR, did PCR again using the new colonies or the isolated plasmid DNA, I got nothing.
Hi again,
The colony PCR is faster and it works for a first screening, but it results in false positives because rest of the ligation products are spread with the bacteria. I agree with Uwe, the best characterization is subculture of the colonies, DNA minipreps and digestion.
And for the digestion of the PCR products it would be easier if you cloned them first in a cloning vector as mentioned above.
Tamara
I agree with Tamara, Colony PCR from the E.coli plate does give you a lot of false positives. They can be reduced by first picking a replika plate and perform colony PCR on those colonies. Or, also preferred by myself: test digest.
Maybe those 3 promoters are quite active in E.coli and there's a selection bias against the final construct, so try picking E.coli colonies of different sizes and screen a lot of colonies.
If you use the same enzymes as for the other 4 constructs, I would not think they are the problem in this case.
Jara
Dear Tamara and Jara,
Thanks a lot.
Before, I actually picked up many many colonies after the transformation and directly transferred them into liquid medium for cultivation. After overnight, I isolated the plasmid DNA and performed PCR. No positive result.
So, I agree with you that the results that from the colony PCR were just false positive results.
It seems that right now the key point is: How to really insert the promoter regions to the vector and avoid the false positive result. Plasmid digestion is a good idea but what should i do if I still cannot find the correct insert?
By the way, the promoter regions that we are dealing with are from P. aeruginosa PAO1, not from E; coli. Also, I think E. coli does not have these promoter regions, otherwise, I will always get positive results by performing colony (Note: After subculturing the cells to new LB plates, I cannot get positive result for the colony PCR).
Dear Jara,
Thanks again for your suggestion. But I am not sure what does the "replika plate" mean (I am not a native English speaker and not major in Biological Sciences). Would you mind explaining this a little more?
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