posted 2 months ago

A potential hack for amplifying AT-rich & repetitive regions

I've been having trouble amplifying a 3kb promoter region that's AT-rich (80%!) w. lots of repetitive elements, then I saw this paper: https://bmcresnotes.biomedcentral.com/articles/10.1186/s13104-017-2982-1.

Gave it a go without much hope, but lowering the extension temp and adding some MgCl2 instantly did the miracle!

I tried both Phusion and Q5, but it seems Q5 didn't work as well - sometimes gave multiple bands when Phusion was still one bright band.

Did not see a significant difference between 1.5 mM vs. 2.5 mM MgCl2, but adding MgCl2 definitely helped.

Tried this method with promoter regions of different genes, and it seemed that the best extension temperature needs to be adjusted a bit - some regions work perfectly at 70C, but others need to be lowered to 67C, for instance.

Hope this helps if you're also having trouble amplifying some difficult regions!

2 months ago

This is awesome!! Did you clone any of these amplicons into a vector? I always have had a lot of trouble cloning in A/T-rich regions even if I can amplify them (or if I just get them synthesized).

2 months ago

Yes I used all the successful ones for cloning! It still varies from gene to gene but the success rate is so much better than before (which is almost 0 lol). Using this PCR method, I did dTOPO cloning and also Zero Blunt TOPO cloning, both of which worked very well. I found sometimes when the primers are slightly longer or have a little bit more mismatches, the success rate also just drops. I wanted to digest+ligate a 3kb promoter into an existing construct, for example, and the 3kb region amplified perfectly with this method. But once I tried primers with restriction sites added, it needed a lot of adjustments and the final PCR product was multiple bands too.

about 1 month ago

I use KODXtreme for everything. I have not tried it with something like your amplicon, but it always works in my hands and I never have to optimize my primers or anything. I also get so much yield that I started scaling down my PCRs and doing fewer cycles, so it is worth the extra $. I would also try lengthening extension times for anything tricky, no reason not too unless you are in a hurry.