Hello, I have been trying to linearize a vector through PCR in prep for cloning for several weeks now. I am using Inverse PCR as opposed to enzyme restriction digestion. However, every time I have run the gel, I do not get any bands. I have tried gradient PCR as well as adding 2% 5% and 10% DMSO by volume. The only thing that seemed to somewhat work was when I added 5% DMSO on top of touchdown PCR. This gave me a band where I expected, but it also showed several other bands as well. The vector is rather large (~6Kb after linearizing, 10Kb before) and the primers had a high GC content which is why I originally added more DMSO. At this point my plan is to redesign the primers, but I would like to avoid it if possible. I am using NEBNext® Ultra™ II Q5® Master Mix instead of Taq polymerase if that helps!
AS well as using dmso try running the pcr with a final concentration of 1molar betaine to help open p the structure of the template dna. If the enzyme comes with a high GC buffer then use this buffer without adding more dmso or betaine, Check the number of copies of template dna. About 30000 is good. It is easy to add too much template when using short dna templates
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