Question of QIAGEN's QIAquick Gel Extraction Kit - (Feb/04/2008 )
I use the QIAquick Gel Extraction Kit of QIAGEN to purify a 350 bp of PCR product from an agarose gel. Unfortunately I do not get anything after gel purification.
I am sure I follow the protocol exactly. And I add the 1 gel volumn of isopropanol after gel dissolving completely. I use 1% agarose gel in 1x TBE. I see very strong band at exact size after PCR.
Where does my DNA go? Any suggestion or comment? Thank you very much.
I use to use (last year..) that kit and worked fine......
I only add 5 additional minutes to get rid of all the alcohol.
Other thing you can do is after disolving the DNa in your buffer you can pass it through the column again.... to recover as much dna as you can.....just get it from the tube, put it in the same column and centrifuge 5 minutes....
good luck
Try adding 10ul of 3M sodium acetate just before adding isopropanol. We always added it while using Qiagen kit.
Thank you all very much. I will redo gel gxtraction today using all the strategies you guys mention. I will keep you updated.
Hmm...interesting. Does that make a difference?
That's what I was thinking too..
Isoprop alone should do the trick.
For all Qiagen kits that must add alcohol step check the viscousity (???) if to much viscouse add a little bit more of alcohol other important step is after final wash centrifuge again to rid any trace of the alcohol. Other thing is that can use a 0.8% agarose gel. For me I prefer to not extract from gel unless extremely necessary...If the product is for sequencing or cloning use other alternative for purification.
Thank you all for the useful inputs. I figured out what is my problem. The yield of gel extraction is kind of low. I can not detect DNA by NanoDrop. After I run it in gel. I do see the band. The concentration of DNA is around 100~200 ng/ul. Usually this kind of concentration can easily be detected by NanoDrop. I do not know why the concentration of DNA after gel extraction can not be read by NanoDrop.
Hmm...interesting. Does that make a difference?
I have never actually compared them side by side. we never used Qiagen gel extraction kit without adding sodium acetate. My previous boss insisted on this step to everyone in the lab.
Hmm...interesting. Does that make a difference?
I have never actually compared them side by side. we never used Qiagen gel extraction kit without adding sodium acetate. My previous boss insisted on this step to everyone in the lab.
I did the comparision. Once the solution is yellow after the gel has been dissolved, there is no difference no matter of the addition of sodium acetate.