How to remove PCR inhibitors from DNA - (Oct/26/2004 )
hello,
does anyone know of a good way to remove pcr inhibitors during DNA extraction. currently we are using a qiagen kit and it appears that there is still inhibitors that are abolishing the pcr's i run 
abolishing? what do you mean?
if you are getting no result, and are using the qiagen kit, i dont see what can be inhibiting the reaction.
what are you doing the pcr ON? genomic dna? (try a touchdown pcr pogram) plasmid? (try lowering the annealing temp)
make sure your primers are good.
so many things can go wrong in pcr. but if you are using kit's i dont see how inhibiters is the answer.
but i might be wrong...
Hi, So many things influence PCR reaction so you can not say there are PCR inhibitors in your DNA samples. Qiagen can clean the DNA or RNA sample very well. I do not think this is a issue. If you have suitable DNA control from any kit, you may try it first to explore the PCR condition for this pair of primers.
Biomed
Hi,
Have you tried just adding some magnesium?
What you describe has happened to me simply because I was solubilizing my maxipreps in TE buffer & was presumably chelating out the magnesium needed for the PCR.
Best of luck!
if you are getting no result, and are using the qiagen kit, i dont see what can be inhibiting the reaction.
what are you doing the pcr ON? genomic dna? (try a touchdown pcr pogram) plasmid? (try lowering the annealing temp)
make sure your primers are good.
so many things can go wrong in pcr. but if you are using kit's i dont see how inhibiters is the answer.
but i might be wrong...
hello,
i have quantified my DNA, spectro and my 260/280 ratio is not that good (using nanodrop), 1.3-1.6. the pcr works in some run and not in others. the controls are fine, but were extracted differently. i was just wondering whether there are better kit or methods of extracting gDNA.
Another very nice choice is the magnetic silica extraction. I've been seen diferent publication related to the miniMAG instrument for 1 to 12 DNA or RNA extractions in only 45 min.
Seems to be a good methodology with a very good performance and the DNA/RNA yield is really good and like a silica method, the pcr inhibitors are avoided. In the September JCM you can find a very good article with this method even using urine, (JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2005, p. 4830–4833).
I would look elsewhere (like primers) for the problem. I mean, many of us (including me) do PCR screening by colony PCR -- a bit of the colony off the plate directly into the PCR mix, and it works fine. That DNA is not purified at all.
clean it up using chargeswitch from invitrogen