I have used the megaprimer PCR approach in the past to make mutations with no problems, however, the mutagenic primers typically mutated only one or two codons and now I want to mutate at least 25 codons. I have designed a primer with (starting from the 5' end) 20 bp of wild-type DNA followed by 75 bp of mutagenic seq. followed by 20 bp of wild-type DNA. The mutagenic primer will be used to make a ~250 bp megaprimer. Has anyone done something similar? It looks straight forward, but you never know until you try.
I have never done this but I talked to people who did this before.
What seems to be promising is to generate a PCR primer pair (by PCR ) and to use that in mutagenesis PCR. So, if you are looking at generating an insert (?) just PCR your fragment together with flanking sequences and use that as PCR primer pair.
I heard about ~800 bp primer pairs before, so you should be good based on size. Just try and optimize.
Also, consider the cost of your labor and the materials and judge if synthesizing that construct might be cheaper and faster.
Hi Stefan,
I want to mutate a 75 bp region of DNA using the megaprimer approach. Have the people you talked with done that? I am not trying to generate an insert.
Is a PCR primer pair of 800 bases the same as a megaprimer of 800 bp?
Hi Pia,
I have not ordered the the primers yet, but I would like to order them today. Doing only half the mutations at a time would be easier, but that would double the time/work and increase the costs.
I agree longer WT flanking sequences would be good, but I am limited to 125 bp for the oligo synthesis.
You can split the 75nt on two primers and copy the vector all way round. All you need is a unique restriction site generated at the 5' end of both primers. just design those primers, PCR around the vector and restriction digest/ligate the PCR product. Add DPN1 in the restriction digestion. Works every time for me.
Hi Stefan,
Yes, inverse PCR would make it easier but then I would have to sequence a large coding insert, and I want to avoid doing that. Adding the two unique RE sites is a possibility and then I would not have to seq. the whole insert "after" the RE sites are in the insert, but initially I would need to seq. the whole insert when I cloned in the insert with the two RE sites.
Thanks
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