Hi, I am new to site directed mutagenesis, my teacher design primers for site directed mutation having TM of 70.14C ,57.03C,53.89C and 59.69C respectively . I have made master mix for my PCR with low TM 53C and high TM 55C, confirmed the amplified PCR product by agarose gel , then digested by dpn1 and transferred into competent cell DH10b , made the single culture colony and then extract the plasmid DNA, Determined the concentration of plasmid DNA by NANODROP and then digest the extracted plasmid DNA by Xho1 and Nde1 to confirm the gene of interest in extracted plasmid. after confirmation the presence of my gene in plasmid I sent the plasmid and bacteria to company for sequencing, but after sequencing results, no mutation occur in two samples while in the remaining two samples mutation occur along with secondary structure annealing ,
Please let me know what should be the possible reason to over come the problem of secondary structure annealing in site directed mutagenesis and what will be the best TM for my PCR to get good result of mutation ?
I will be extremely thankful for your help
Hi, it seems from your question that you were using one-step PCR mutagenesis, amplifying the whole plasmid with mutagenesis overlapping primers and using PCR products directly for transformation after DnpI digest to get rid of methylated template. If that's the case, I found that sometimes I needed to sequence DNA from quite a few colonies to get one with mutation. I think that it could be due to too high concentration of DNA template in PCR reaction and inefficient DpnI digest. I would try to lower DNA template concentration or use an alternative cloning strategy with overlap PCR.
Hi,
Before jump to the conclusion that secondary structure was the suspect for your result I have 2 questions that might help you find out the problem and/or solution.
1. How do you sure that your digestion with DpnI was succeeded? after digestion with DpnI you didn't explain whether you insert it to a carrier plasmid before transform it to the DH10b, did you just transform that pcr product anyway without inserted it into such carrier plasmid with amplification origin sequence?
2. I am curious with what was the template you used for PCR? If you are using plasmid carrying your GOI as a template for PCR, I wonder if the single colony you've picked was the plasmid you used as template. not that plasmid carrying your amplified gene. That is why no mutation in your gene, because it might be derived from the original plasmid. If I were you, I will not continue to plasmid purification anyway if I only found single colony.
Cheers,
Kurnia
Sure you test every single step before going to the second step. Confirm that you obtained the mutated sequences from your PCR, ligate it to your plasmid, and then proceed with your transformation. You have to compare the digested and undigested plasmids on the same gel if your confirmatory test is a restriction digest or alternatively do the sequencing. If from the sequencing you can find the clone of interest, then you can work on this clone and proceed with further PCR optimizations.
Hi, it seems from your question that you were using one-step PCR mutagenesis, amplifying the whole plasmid with mutagenesis overlapping primers and using PCR products directly for transformation after DnpI digest to get rid of methylated template. If that's the case, I found that sometimes I needed to sequence DNA from quite a few colonies to get one with mutation. I think that it could be due to too high concentration of DNA template in PCR reaction and inefficient DpnI digest. I would try to lower DNA template concentration or use an alternative cloning strategy with overlap PCR.
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