point mutation - (Jan/21/2008 )
I used the PCR method to make the point mutation in a plamid of 17kb. After transformation, there are several bacterial colonies growing up. But after PCR assay and restriction digestion assay, it is not what I want. Normally, if the mutation has not been made out, it should be the original plasmid which has not been digested completely by DpnI . But they are not the original plasmid too. Then what are they?
Hope anyone can help. Thanks.
I would guess some sort of recombination events is taking place which is why you have plasmids different from the original. What cells have you used for transforming them? You make have to use some other cells for your transformations like SURE or Stbl3 cells.
Thank you, scolix!
What do you mean the recombination events? Which stage can it happen? In PCR? In digestion? or in transformation?
I used the DH5a competent cells. Is it OK for the mutation transformation experiment?
I have another question. After PCR and digestion, if I do the pheno-chloroform ethanol ppt to treat the sample, does this treatment damage the mutated plasmid containing staggered nicks?
Thank you.
What do you mean the recombination events? Which stage can it happen? In PCR? In digestion? or in transformation?
I used the DH5a competent cells. Is it OK for the mutation transformation experiment?
I have another question. After PCR and digestion, if I do the pheno-chloroform ethanol ppt to treat the sample, does this treatment damage the mutated plasmid containing staggered nicks?
Thank you.
It can happen in the bacteria after transformation. If its recombination, you can avoid it by using the bacteria which can prevent recombination.
I don't think it should be a problem to ppt after the pcr and digestion. We usually transform cells after digestion without any ppt or purification for mutagenesis.
Thank you!
Is it more difficult to make a point mutation in a large plasmid of 17kb than in a small one of 9kb? I used the expand long template PCR system instead of using the Pfu polymerase, is it OK?
In addition, i used the NEB dpnI to digest the template plamid instead of stratagene's dpnI , does it work good?
Is it more difficult to make a point mutation in a large plasmid of 17kb than in a small one of 9kb? I used the expand long template PCR system instead of using the Pfu polymerase, is it OK?
In addition, i used the NEB dpnI to digest the template plamid instead of stratagene's dpnI , does it work good?
i am not sure how well the mutagenesis works in 17kb plasmid compared to smaller ones. Most of the ones we have made here are less than 9 kb. We have only used PfuI, so not sure about other enzymes.
We use Neb's dpn I instead of stratagene. Just use the same amount of units. It works the same.
Thanks!
I have a question about the mutant strand synthesis. The dsDNA template recommended ranges from 5-50ng, why is the restriction of the amount of the template? What king of result will be led to by excess template?
I have a question about the mutant strand synthesis. The dsDNA template recommended ranges from 5-50ng, why is the restriction of the amount of the template? What king of result will be led to by excess template?
I think, having low amounts of template reduces chances of mutations other than the desired ones. With excess template you might have unwanted mutations.
Thank you