QuickChange primers self-made - (Jan/23/2007 )

Hello everybody,

I hope this question hasn't been asked before. If it has, I am sorry for bringin it up again. I did not find an answer to it in any of the posts in this forum.

My question is: Is it possible to use self-made primers for QuickChange mutagenesis? The primers I need for the mutation are very long. Therefore I would like to make them myself with PCR, purify the product from agarose gel and use it as the QuickChange primer. Has anyone done this before? Or does anyone know, why this might not work?

Thanks in advance for your reply.

---

I think this should work. You'd want to use a proof reading PCR enzyme to avoid A tailing. The required concentration of DNA is pretty high, but easily achievable. How are you planning on putting mutations into your "primer" PCR reaction? Normally there would be problems in that the PCR product is double stranded, but here that is exactly what you want in any case.

---

I planned to use Vent polymerase. I think this is proofreading, right?




Actually, what I want to do is an insertion. So I would do a PCR on the segment that I want to insert, but with primers carrying at the 5' a complementary sequence to the plasmid, where I want to insert the fragtment.


Thanks for sharing your opinion on that.

---

Well, I wanted to let anyone who is interested know, that this strategy of making your own QuickChange primers by PCR works perfect. Like this I then usually order two short (~20 nucl.) primers with complementarity to the insertion site in the plasmid at the 5' and with complementarity to sequence to be inserted at the 3' end. Then I run a normal Taq (or Vent) PCR, purify the fragments with a PCR fragment purification kit and use the eluted double stranded fragments (which usually are around 0.5-1 uM) as primers in a QuickChange reaction. Saves you a lot of money, since less longer primers have to be bought, which for this reason also do not require any special purification (plain desalted always worked for me).

Cheers.

---

How about just ordering a longer primer for 30-40 bases, with no special purification. Its a tiny bit more expensive than 20 bp primer and one doesnt have to go thru the PCR and purification before the mutagenesis step?

---