faint pcr bands to some, intense to some - (Nov/12/2005 )
another question. I used equal concentrations of dna sample ( 100ng/ul- with less than 1.6 purity) for my multiplex PCR. MY question is:
1. should i expect that i get same band intensities considering i used equal concentration of pcr mix and template in all samples?
2. if not, what are reasons for showing faint bands for some and intense for some, considering i have equl dna concetration as template?
thanks all again
Multiplex PCR can be really difficult. It is very sensitive to Mg++ concentration, and some primer combinations just don't work no matter what.
So if you are referring to a comparison between different primer sets, it is not surprising at all that you see the different intensities.
well ur question seems to unclear, are u seeing bands of different intensities in the same lane, as multiplex pcr is used to simultaneously amplify two targets......or is it that u have used two different primer sets for the same template.............i hope u got it!!
hi again. Oh am sorry for not making that clear. I was refering to different band intensities of the pcr product of one lane as compared with the other lane ( two different DNA samples from two different cases). But i used same concentration of dna template and same concentrations of primers etc.
The intrnal control for example is intense in sample 1 but faint in sample 2, considering i used the same DNA template concenration and pcr conditions and mastermix. Is it common?
A couple of comments:
1) There may be varying inter-sample amount of interfering substances in your DNA prep.
2) Are you sure of your DNA concentration? (e.g. phenol or guanidine may interfere with spectrometrix quantification). A 260/280 ratio lesser than 1.6 looks bad to me.
3) Is DNA partly degraded (possible, if unlikely, source of error)
I've tryed genotyping using multiplex PCR (trying to see at the same time the WT allele and the KO one in the same heterozygous animal). Well, it was impossible to reproduce the results, which varied from nothing in the lane, just the KO one (smaller and probably more efficient) to both of them but WT very faint. So... I gave it up and went for separate pairs of primers in separate reactions. You might want to try this.
What template are you using, genomic? Because genomic DNA is notorious for strange behaviours in PCRs, or at least that's my knowledge...
A lot of things could go wrong: primer-dimers, all sorts of competitions, hard to access loci in genome, etc.