Mutagenesis using 4 sets of primer - (Jul/05/2006 )
hello to all PCR experts
I am planning to do site directed mutagenesis by using 4 primer sets two having restriction sites and two with change nucleotide . but i have some query::::
1) i am confused about that after getting two seprate PCR how i should proceed to get one single product.
2) same thing i am also trying by using DpnI and pfu polymerase ( total size 3.5Kb vector + 1.3Kb DNA fragments) but on agrose gel nothing is vissible at 4.8kb but DNA stuck at well only. should i proceed it for DpnI digestion as in stratagene manual it is written that some time in gel u will be not able to see anything. after DpnI digestion XL1Blue is only option or i can transform it in any competent cell.
thanks in advance
I didn't understand very well what you want to do. I think the right way to proceede would be to do the first QC, DpnI digestion, purification of the PCR product, quantification of the product and second QC with a tiny amount of template. But your template will not be methilated, therefore you cannot get rid of it with DpnI. Cannot you design the primers with the mutation and the restriction site together?
Otherwise you can transform the first amplified DNA after DpnI digestion and then use the DNA you obtain from some colonies as template for the 2nd QC. A lot of work...
And yes, you can transform any competent cells
actually i am planning my work like this which is more clear in picture attached with this mail.
and my second query that before dpnI digestion u will be able to see ur PCR fragment in gl or not as in my case some DNA stuck at well only.
thanks
It looks quite complicated and I don't know if you can do it like that... Wouldn't it be easier to do the first amplification and then the ligation in a vector directly?
its posiible only if i will do amplification of PCR product with M1 and M2 as it is already cloned in Pet 23a, so i will get full fragment including vector seq. i did it but again after dpnI digestion i have to transform it XL1blue or simple DH5a is ok????. if u plz tell me in little detail as i am new in molecular biology.
thanks for ur suggestion.
You can use any strain, as long as it is recombinase-. DH5alpha is fine. And I use DH5alpha or DB101.
Where are your restriction sites? Wheer is your aa change? Can you indicate the primers as you did in the figure?
I don't see why DpnI digest shouldn't be visible on the gel... I think maybe it's more difficult to see the DNA because some of the molecules are ssDNA because you digest the parental molecule they are complementary to.
Where are your restriction sites? Wheer is your aa change? Can you indicate the primers as you did in the figure?
I don't see why DpnI digest shouldn't be visible on the gel... I think maybe it's more difficult to see the DNA because some of the molecules are ssDNA because you digest the parental molecule they are complementary to.
Hello,
I think what you are attempting to do is a fusion PCR where you start off with two fragments which have a complementary sequence to each other. I am also trying this with little success at the moment. From what I have read in the literature people tend to have an annealing step prior to the 3rd PCR. The conditions for this annealing step vary greatly in the literature. I have to anneal a 800bp fragment to a 2700 bp fragment but I always end up seeing just the same two bands after any annealing step I use.
