DNA purification from agarose gels - (Nov/30/2005 )
I am trying to subclone a 17 kb DNA fragment from a BAC clone into the pBLU2KSP vector. I am able to digest the fragment (EcoRI) and can see a bright band when I run the digestion products on the gel. However I have had very little success in purifying that 17kb fragment and the DNA concentration I am getting is not enough to reach a 3:1 molar ratio with the vector. I have tried the QIAEX II gel extraction kit from Qiagen as well as the ultraclean gel spin DNA purification kit from MO BIO labs with no luck.
I would appreciate any input on this issue.
It is very possible that your 17 kB fragment is too large to bind to the column and is in the flow thru. I would put it thru the qiagen mini-prep kit.
A student in the lab sucessfully purified a 16 kb plasmid that way... it might work for your fragment. Be sure to use the EB buffer, not water.
Melt your gel and apply it to the column.. see what happens.
also try to warm the elution buffer ... in our kits (molzym) it's recommended to use 70°C elution buffer if DNA is larger than 10kb