Elution - elution problem (Jun/04/2007 )
Hello iam facing problem in gel elution initailly my band is thick intensed i dissolved finally in 5o microlitres but still i loaded 5 microlitres of sample but no band iam doing this with Qiagen elution kit loosing a lot
please suggest me anykit/method to get good elution in PCR product
i use Easy Trap ver. 2 from TAKARA for gel elution and i never faced any problem. i get high yield, and its very easy and take 20 to 30 mins only
how much DNA do you estimate was the starting material and compared to that how much could have been there in 5ul of the eluted solution if you lose 10% of the initial amount.
If you use 1 full Qiagen kit, there might be 1-2 columns defective where you can lose most of the DNA. But this is not very likely. Invitrogen is also quite good.
Before you elute your DNA with the provided elution buffer, try heating it in an oven or a heat block at 55 degree Celcius, 5 to 10 min. Then spin to elute. You won't get 100 % of the initial input though.
you'll definately lost some of your sample in any column based extraction/purification kit. a friend in my neighbour lab also facing the same problem. but after she told her problem to QIAgen and they substitute another box for her (free, of course!), her problem solved. why not you tell the representative from QIAgen about this and let them troubleshoot for you (try it out for you)? they'll know better.
or you might want to check if anything (tips or tubes) that you used has DNase? are they all DNase free?
or you leave your gel outside/under UV too long before you cut and purified?
or try to elute in less volume (20 - 30 microliters)?
For me, i never have any problem with QIAgen product. my DNA is extracted everytime i extract, never fail, not even once.