I am doing a mutation in a 80% approx. GC content region of my cDNA in a plasmid. I am using the Agilent QuikChange XL II mutagenesis kit. I've used primers designed by the Agilent's software (complementary ones) that are 23 bp long. The first time it did not work. I tried to use a 7% DMSO and it does not work as well.
Now I've designed new primers that are complementary in only the mutation region and 5' extended (50 bp in total). What do you think? Do you have any more clues to solve this problem?
Thank you.
https://www.researchgate.net/post/When_designing_mutagenesis_primers_what_is_the_difference_between_those_that_anneal_back-to-back_compared_to_those_that_have_overlapping_5_ends
You can try sequencing your cDNA that has mutations using forward primer upstream to the GC rich mutated regions and design reverse primer in the downstream region. Later after after getting the sequencing data design appropriate primers targeting your mutated DNA region.
Hope your experiment works.
What do you define as it not working? Did you not get any colonies? Did you have a lot of colonies that all came back as your WT cDNA?
Have you tried other additives like 0-10% Ethylene Glycol or 1M Betaine instead of DMSO?
Also, while the Aligent software designed only 23 nt primers, I would suggest making your own primers that extend 15 bp both 5' and 3' of your mutation regardless of what the Tm is calculated. I also suggest extending your extension time by at least 1 extra kb of DNA (if your plasmid is 6 kb and your polymerase is 1 kb/min, I would use a 7 min extension time over a 6 min extension).
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