purifying small DNA fragmets - (Aug/03/2008 )
I've posted this in the real time PCR forum without many answers, so I'm cross posting to see if anyone else has a suggestion.,
I'm trying to clean up my DNA (two fragments around 100 bg, one around 150 bp) to make standards for real time PCR. 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 and probably will not digest out the Sybr. There is one strong band, so it might be ok, except for the Sybr.
I have been recommended to use either CentriSep columns or Illustra GFX PCR and Gel Band Purification kit
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?
3/ Any experience with the above kits
Thankyou for your help, oh wise ones
I have used invitrogen's gel purification kit to purify 110bps of pcr fragment. It worked quite effectively for me.
good luck !!!
Same thing, I use GE LifeSciences GFX columns to purify fragments as small as 50 bp, always good yield.