Gel extraction help.... desperate! - (Jun/09/2009 )
Hi,
I've just done my nested PCR for 5' RACE and cut out the band that I'm interested in (~1 - 1.2 Kb) from my gel and want to gel extract. The band isn't very bright but the kit that I bought (Invitrogen GeneRacer kit) had spin column that claims to work for gel purification that would give you purified product that can be cloned/sequenced. Tried and ran on gel again to see if I have the band I want and nadda. I've tried Qiagen gel extraction kit as well and also nothing.... Any ideas on what I can do? Is it because I didn't have enough product to start off with so I'm just losing everything when I elute from the columns? Or am I doing something very very wrong?
It's possible you have recovered enough sample to clone, but not to see on an EtBr-stained agarose gel. Try the cloning.
Try the cloning. That's what I would say too.
If that doesn't work:
Try to optimize the PCR o get more product, or elute without using a kit (methods using filter tips or freeze-and-squeeze are explained elsewhere in the forum).
mastermi on Jun 9 2009, 04:39 PM said:
If that doesn't work:
Try to optimize the PCR o get more product, or elute without using a kit (methods using filter tips or freeze-and-squeeze are explained elsewhere in the forum).
I agree with my colleagues and suggest you go ahead with the cloning.
If you absolutely want to do gel purification do it the old school way,do a phenol extraction, run a really low melt gel around .6 or 0.7%. it gives far better yields than the qiagen kit anyday, atleast in my hands
hope that helps
good luck
moerae on Jun 9 2009, 02:15 AM said:
I've just done my nested PCR for 5' RACE and cut out the band that I'm interested in (~1 - 1.2 Kb) from my gel and want to gel extract. The band isn't very bright but the kit that I bought (Invitrogen GeneRacer kit) had spin column that claims to work for gel purification that would give you purified product that can be cloned/sequenced. Tried and ran on gel again to see if I have the band I want and nadda. I've tried Qiagen gel extraction kit as well and also nothing.... Any ideas on what I can do? Is it because I didn't have enough product to start off with so I'm just losing everything when I elute from the columns? Or am I doing something very very wrong?
I gel extract quite often and have had great luck with the MoBio Gel Extraction Kit (all their stuff has been pretty solid)... Qiagen kits have always worked ok, but they have a reputation for being really inefficient. You can also try to concentrate the DNA solution after gel extraction with a speedvac or something like that. Using less volume in the final elution may also help (30ul instead of 50ul), and or try to let the elution solution sit on the spin column for a few minutes before centrifuging.
Like everyone says - go ahead and clone. I use the Qiagen gel extraction kit and routinely get negative concentrations of DNA on our Nanodrop, but still get colonies when I clone.
I don't like Qiagen's kit, usually Fermentas' gel-purification kit(silica-based) gives me better results.
