5 replies to this topic

[Curtis]

I am designing primers to produce two PCR products that have an overlapping region, and then run Overlapping-Extension-PCR with these PCR products as template.

Some people say keep the overlapping size at 40-45 bp, but this way the annealing temperature will be very high (nearly 81C). I want to keep it at 21 bp, meaning desining two primers that are exactly complementary of each other (Reverse primer of Fragment 1 to be complementary to Forward primer or fragment 2). should I worry about the length at all? or they will bind anyway?

Edited by Curtis, 09 April 2012 - 10:04 PM.

[pcrman]

A overlapping length that is the typical size of a primer should be fine. You can design the middle primers (both forward and reverse) at the same location.

[Curtis]

Thanks, so the size doesn't matter

However I already designed primers for both strategies, let's see which one works.

[Curtis]

just to update this thread.

I tried the OE-PCR two times, it didn't work. it gives me a strong smear above my expected band size. It could be because of long extension time or high concentration of template DNA I think. fragment 1 is 3800 bp, fragment 2 is 3000 bp. all primers have Tm of 59C. I use phusion polymerase.

The overlap region is 44 bp, but I still had the annealing temp at 59C for the first 10 cycles. I think it is low. I need to increase it. for the 2nd set of cycles (30x) I will go back to Tm 59C.

some people also suggest taking 5 ul of the 10-cycle reaction into a fresh tube with the F and R primers (instead of just adding the primers to the used reaction).

[Curtis]

I can't get this done, I'm really down.

[Curtis]

Hi all, I just want to update this topic. I finally managed to get my OE PCR product according to what pcrman suggested. I kept the overlap region at 23 bp, and used phusion polymerase from Finnzyme, although some guy at wikipedia has written to not use phusion polymerase. I even contacted him, and he kept insiting I should avoid phusion. But I got the product anyway. I fused two 3.3 kb fragments to each other to get a 6.6 kb product. My annealing temp was around 61C. I also noticed products in DMSO amplify better.

So thank you pcrman.

In a separate expriment I cloned the 3.3 kb fragments into pJet and ran OE with those. The reason was to use more template. After PCR I added DpnI to my PCR reaction for 1 hr. When I ran it on the gel there was no PCR product. It is like if the fragments only fuse when they are not cloned. I am not sure why.