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
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.
how about your template?
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.
i used new batch of all my ingredients (buffer etc) and it worked nicely again.
now i alliquot all ingredients (except Taq) in small volume. i hope it can work nicely in longer time.