problems cleaning up small DNA products - (Jul/31/2008 )
This real time PCR is a lark isn't it!
I'm trying to clean up my DNA (two fragments around 100 bg, one around 150 bp) to make my standards. I've been using the Ultraclean Gelspin kit.
I get great bands on a gel after PCR, then after either digesting the agarose as per the kit, or purifying the straight product as per kit I get terrible yields according the the spectrophotometer. When I run this out on a gel with hyperladder, there is very little there too.
So lurking around on the forum has yielded the information that small DNA fragments can get washed straight through the column. When I checked my kit, it said that it can do 100 bp-50 kb, so I am on the low end for that kit.
I suspect I've been washing the fragments through, as it is the purification step which is getting rid of my lovely DNA.
My post doc has some ExoSAP but I'm not sure whether using this will lead to pure enough DNA to quantify by spec for the making of standards. I don't have appreciable primer dimers on a gel, but still..
1/ can I use ExoSAP to purify DNA to make real time PCR standards
2/ if not, which kit or method will reliably deal with 100 bp fragments?
Thankyou for your help, oh wise ones.
this is a bit old fashion, but you can use electrophoresis followed by electrolution to purify the DNA fragment. Dialysis tube can retained a 100bp fragment (if you don't have an electrolution tank and have to use a standard electrophoresis tank)
Going to answer my own question here, so if anyone is looking in the future, they have an answer.
I've used the illustra GFX™ 96 PCR Purification Kit from GE Healthcare which purifies down to 50 bp. We've added in an extra spin for 30 seconds prior to elution and used 50 ul to elute.
So for the first time I've got good bands on a gel in comparison to hyperladder after a clean up of small PCR products. Now for the nanodrop to quantify.