GC rich Exon 1 in ATP2A2 gene - Anyone heard of betaine? (Jun/01/2006 )
Greetings to everyone on the board,
I've been trying amplify exon 1 in the gene ATP2A2. It's the 1st exon in the gene so it was a high GC rich content. I'm not getting any bands, thus no amplification. I've tried using the Fastart GC Rich Kit. But I've had no luck.
However, I found a paper that also amplified the same exon! They used Betaine in addition to their standard PCR mixture. This is new to me. Can anyone suggest a how I can get 2 M Betaine? Or another way to amplify this GC rich region? Thanks.
Can anyone suggest a how I can get 2 M Betaine? Or another way to amplify this GC rich region? Thanks.
Betaine is a fairly standard reagent from Sigma, and others.
You could try using DMSO as an alternative (1-5% in the reaction mix).
Keep the primers short (to give you a reasonable Tm ~65C) unless the sequence has lots of degeneracy.
Do a touchdown PCR. This should help with mispriming events.
Apart from that, use the standard PCR aids...Wear your lucky socks / T-shirt / whatever.
Thanks for the reply Swanny,
Do you know how betaine works exactly??? But more importantly, do you know how much betaine should be added to 50uL reaction tube when doing PCR (asssuming the stock solution is 2M)? Thanks for your input...I'm really new at this.
betaine is sometimes known as Q solution......fyi
it disrupts secondary structure...
i usually use around 10 ul in a 50 ul reaction; but you can titrate this for your own requirements.....