QIAquick PCR for preparing for restriction digestion - very low concentrations (Apr/11/2006 )
I am preparing to clone 4 fragments between length 350 to 75 bp into a pGEM 3z vector
for this I am PCR amplifyging these fragments from a construct in the lab
I get very good PCR bands for 100uL reactions
After thie PCR step I tried to purify the PCR products using QIAquick PCR purirfication kit
I got very low yields of DNA when i used 55 uL of water for elution..
the maximum total amount that I have is 2.5 ug
this further needs to be digested using bamHI and ecoRi and then again purified for ligation
do u think i have enuff for ligation
if not what mistakes could I be making
the consensus among many of us (myself included) seems to be that if you are using that kit, it vastly increases your yield if you use elution buffer warmed to 55-60C, and let it sit on the column for ~5 minutes before spinning
i agree with aimikins, but will add, check the pH of your water - you may get a better yield if you have the pH around 8 or use the 10mM Tris EB....
I agree with the two guies upstairs.
Add one more point, what I usually do is to use the QIAEX gel extraction kit (does not use the column) instead of QIAquick kit.
My experience that the QIAquick PCR purification is good for such sized fragments, I even used it for fragments between lengths from 70 to 60 bps. Furthermore, I’d feel that you have enough purified products for ligation take consideration of the products’ size. If you like, you might also reload elution to column and spin again for higher recovery (most time it is no necessary).
Have you run gel check purified products? Some time I do not believe concentrate estimated based on value of OD from UV Spectrophoresis. I have experience with no purified products based on value of OD but a strong band in agrose gel elecrophoresis…