I am using recombination cloning and using in home prepared DH5a cells , the efficiency of cloning is low (~40%or less).
For screening my clones for insert using PCR, I am using universal plasmid-based primers, and I tried home-made Taq and oneTaq (NEB) for high GC content inserts, none gave consistent results in being able to amplify the different insert. For larger inserts (1-2.5 KB) there is always difficulty with amplifications.
Any help would be really appreciated as I have been struggling with these problems for a while.
You may need to invest in the new Q5 polymerase. It's way superior in terms of long high G/C amplification. Also get some of the OneTaq G/C enhancer to go with it, and try different concentrations.
Cloning is of course a different story. That's where high G/C really makes your life into hell.
I actually tried Q5 polymerase. It worked well with the shorter sequences (
Hi, if you have plamid/insert rearrangement afer cloning, you may want to use the DH10B strain. Bacterial Artficial Chromosomes which contain 200-300 kb of genomic DNA are cloned in DH10B. Alternatively or in combination, you may try to clone your inserts in a low copy number plasmid (e.g.pbr322). Inserts are more stable.
Emilio, Thanks for your answer, I checked the NEB website, it seems the efficiency of the cells is higher than DH5a especially with larger plasmids (almost double the effieciency), my plasmid is 3.5 kb and and insert 2.5Kb is added , 6 kb will result. However, They say that it is unsuitable for unstable inserts. Are GC rich genes unstable? !
Another thing I am thinking of is the use of Stbl2™ Competent Cells from thermofisher as the website says it is suitable for high GC genes. Has anybody tried it or had an experience with it?
Hi Nadia, I do not have experience with the Stbl2 cells, but with the DH10b we were able to clone relative large plasmids (20 kb) with plenty of repetitive sequences. it may also help to culture the bacteria harbouring the plasmids at 30oC or RT,
stbl2 and DH10B both work fine with high GC content cloned genes.
Hey Nadia,
You probably already discovered a solution, but in the outside chance that you din't find a solution, adding a small amount of DMSO to your PCR rxn mixture has really helped me in the past when cloning GC-rich fragments of DNA. I don't remember the exact amount (1 uL/25 uL of rxn mixture or maybe 1% DMSO, I don't know), but if you look online you will discover more information about it. It's much cheaper than buying somewhat pricey rxn components.
Best,
David
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