4 replies to this topic

[ocean]

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  

[Zaax]

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...

[biomed]

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

[JEB]

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!

[ocean]

Zaax, on Oct 26 2004, 12:48 AM, said:

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...

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.