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!
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).