We've tried several kits and they all seem to be hit or miss. We need something that is going to work 100% of the time and with decent efficiency. Does phenol work? Why is this giving us so much trouble?
Hi Jonas,
I've always had issues with agarose gel extractions too. I don't have a magic solution to offer but some minor adjustments would likely increase your yield.
When dealing with PCR bands, I tend to multiplex the PCR reaction. I may pool up to 10 PCR reactions together and precipitate the PCR products with EtOH/acetate overnight. I then suspend the pellet in a minimal volume of water/loading dye before loading the gel. This trick has allowed me to fit the equivalent of 10 wells of DNA in a single one, thus minimizing the amount of agarose at the purification step.
Our lab switched from the BioBasic kit to a Qiagen kit, which was somewhat better. Heating the water/elution buffer may help a little. Try to minimize the amount of agarose by refining your well molding and gel cutting skills.
Best of luck,
Jonas, I am happy to show you what we do in my lab. Stop by some time. It works 100% of the time. Our application is for direct Sanger sequencing or cloning of the bands isolated from the DNA gel.
Thanks to both of you for responding. I know it's an old and common complaint, but I just wanted to see what answers I got on here. Matt, maybe me and my postdoc can come by next week and get some advice. We've tried both types of Qiagen kit and kits from Denville and now Thermo. We usually load 100 uL of PCR product and have only been getting good results about 70% of the time. For cloning we usually just do freeze and squeeze, but we need to process a bunch of these in the near future for Sanger sequencing and it would be nice if you could show us how to bat 1000.
Low density, agarose gels 1%-ish in TAE buffer, low melting temperature , with the smallest amount of EtBR used for visualization possible, and just enough UV exposer to quickly excise the the fragment. Any column based gel purification kit works great. At the time this was routine in my lab we used a kit from Promega. After the band is purified, then do your DNA purification and elute in sterile deionized water. This purification is necessary because TAE has Taq-Pol inhibitors, this applies if you are going to use a "sticky-end" ligation strategy and need to use Taq-Pol incubation to Adenylate your fragment for ligation.
Hi Jonas,
one kit is works better than others is Fermentase gel extraction kit with suspension silicate, columns kits usually does not work well but silicate suspension works better.
I use the Gel Recovery kit from Zymo Research. I've been using it since grad school and won't use anything else. In my mind it's the best in the market and works with low sample volumes for re-elution.
Hi,
Why you cannot purify the PCR product directly after PCR reaction?
Check the presence of the PCR product on a gel, and the rest of the PCR product clean with for instance: PCR / DNA Clean-Up Purification Kit (EURx).
Good luck!
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