I'm cloning a fragment of 3200 nts into plasmid. The cloning was successful, however, 02 amino acids were mutated. Now I want to fix these 02 aa by site-directed mutagenesis technique using original DNA plasmid as template for PCR. The PCR product contains 02 kinds of DNA which are in the same length (original template and newly synthesized product). PCR product will be treated by DpnI to digest all the original DNA. The remain PCR product (my target DNA, linearized structure) will be purified and performed in-fusion to circularized into plasmid, then transformed to E. coli for propagation. Plasmid will be extracted from the E. coli and confirmed by NGS. I repeat some experiments, unfortunately, it seems original DNA was still partly remain after DpnI or the site-directed mutagenesis reaction was not successful (I think) making new plasmid still identical to the original template.
Now I want to check whether my target amino acid is fixed or not before sending sample to NGS optimizing cost benefit.
You don't need to do NGS. Just do a regular capillary electrophoresis DNA sequencing. All you need to do is to order an antisense forward primer complementary to the sequence region 50 bps prior to the point mutation site.
Good luck.
I concur that you only need to do regular sequencing not NGS and this should much much less expensive. However if your clone picked up two mutations during the cloning, why not just look for another clone? That might be easier than doing two rounds of Site directed mutagenesis.
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