4 replies to this topic

[wizzkid]

i was asked by an examiner "How to better optimise my PCR reaction":
I replied change annealing temp (if no gradient PCR was performed), using a high fidelity Taq, applying a longer elongation period at end and  increasing or maybe decreasing pcr cycles depending on the results that where being obtained
He just replied with you need to do more homework    
anyone else have different answers?

[almost a doctor]

wizzkid, on Mar 19 2010, 05:03 PM, said:

i was asked by an examiner "How to better optimise my PCR reaction":
I replied change annealing temp (if no gradient PCR was performed), using a high fidelity Taq, applying a longer elongation period at end and  increasing or maybe decreasing pcr cycles depending on the results that where being obtained
He just replied with you need to do more homework    
anyone else have different answers?

Here the ones I can think of:

adjust Mg2+ concentration (whether its MgCl2 or MgSO4)
adjust primer concentration
adjust template concentration
adjust dNTPs concentration (although this are usually in excess, if this increase you definitely need increasing Mg2+)
consider using additives if needed:  DMSO, betaine... (they're usually used if high GC content, in some of my applications I find them useful to reduce primer dimer)
adjust annealing temperature and time
adjust elongation times (longer or shorter depending on desired product size, ~1min/Kb)

hope it helps!

[perneseblue]

-if AT content is high, consider reducing extension temperature ~68C
-add BSA to reduce amount of polymerase absorbed on surface of PCR tube. (thus increasing effective polymerase concentration)
-change to proof reading polymerase eg KOD, Pfu,
-change concentration of K+ ions.
-dilute template 1:20 to 1:50. Template may carry PCR inhibitor is not completely cleaned. Dilution will dilute out inhibitor, and PCR reaction will still be able to amplify DNA.
- for long range PCR add Uracil-DNA Glycosylase to remove any UTP that might be form from thermal deamination. Insertion of Uracil into DNA causes polymerase pausing, which increases the likelihood of the polymerase dissociating from the DNA strand yielding a truncated DNA product.

[wizzkid]

cheers guys its extended my knowledge at least if anyting

[Denny]

wizzkid, on Mar 21 2010, 08:42 AM, said:

cheers guys its extended my knowledge at least if anyting

Hey Wiz,
My favorite dumb saying is "I don't know what I don't know, until I know it."  

Just becoming aware of what we need to be aware of when troubleshooting is the key and is our struggle while learning new skills. It's the way of things.

Edited by Denny, 21 March 2010 - 04:49 AM.