Protocol Online logo
Top : Forum Archives: : Molecular Biology

Reliability of PCR? - (Mar/17/2006 )

Hi guys, i really need ur suggestions
last week i`ve already found the best PCR condition and got good results (yay!)in my multiplex PCR after million of trials before. but, these days when I repeated the PCR with exactly same conditions for every component (pcr ingredients and thermal cycler), I never got the same bands again. I already checked and checked and...checked my note again to find what`s wrong...but it seems nothing`s wrong....
Any suggestion wil be veeery appreciated
Thanks

(sigh)... sad.gif

-audrey-

Are they still the same PCR components? If so then maybe you need to buy fresh ones. Things degrade over time, even primers and probes.

-Doc_Martin-

how about your template?

-mdfenko-

I find PCR to be fairly consistent as long as your template and ingredients are "happy."

More often than not, I find people to be the limiting factor in PCR as opposed to the technique itself.

-Matt

-MisticMatt-

THANKS!
i used new batch of all my ingredients (buffer etc) and it worked nicely again.
Phhew...
now i alliquot all ingredients (except Taq) in small volume. i hope it can work nicely in longer time.

-audrey-