qPCR

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elsa
elsa's picture
qPCR

Why am I getting a pellet after the quantitative PCR using Sybr green? I'm using a lightcycler (Roche) and this is hapenning with different sets of primers and different magnesium concentrations, and of course is interfering in the results! Can anyone help me?!

nin1318
nin1318's picture
hm i have used sybr green

hm i have used sybr green quite a few times and have never had that happen, nor have i heard of anyone else having that problem...perhaps you could post exactly what you are putting in each well before you run the reaction.

elsa
elsa's picture
yes, this is definetly very

yes, this is definetly very strange...
I'm using the kit from Roche (lightcycler faststart DNA master SYBR green I), so I just add the components of the kit + 2ul cDNA + 2 ul each primer + x ul MgCl2 to get the final concentration required. First I thought that it might be the MgCl2, but I tried adding none (which means 1 mM Mg because the kit already contains it) and it still happens. Then I tried reactions without cDNA, or without one of the primers, and it doesn't happen. Is it possible that I'm using too much primers? or is something wrong with the cDNA I'm using? (the reactions seem to work well on normal PCR!).
Thanks!!!

ToNoFo
ToNoFo's picture
confirm that it is actually

confirm that it is actually the amplification causing the pellet. (1) Run a reaction without primers and see if you still notice it. (2) Also run just a straight master mix with primers only. It is possible that the heating and cooling could react with something in the way you have prepared your samples. Check the purity of your cDNA if (1) comes out negative. If (2) comes out positive then switch your mastermix...