DNA amount calculation for PCR - (Feb/17/2011 )
I was never able to use the eqations like V1C1 something, seems to me too alien, and complicated. I, instead, imagine amounts od stuff flowing in the different volumes like this:
In PCR you work with microliters and nanograms, so let's transform it to them first.
48 microgrammes/mL = 48 nanograms/microliter
that's easy, you lower the volume 1000 times, so you lower the weight 1000 times, that means only to change the units on both sides.
Now what you need in your PCR reaction.. 1 microgram/100 microliters, that's 1000 nanograms/100 microliters -> 300ng/30 microliters, but you choose to use 200ng in reaction insted (personaly I don't think it's that important to calculate the "right" amount for a reaction volume, I just put 100 ng per reaction in normal volumes (10-50 ul) and don't calculate anything)
So you need 200ng in reaction and got 48ng in one microliter, so how many microliters you need to put in reaction to have 200ng flowing in there? You just divide 200 by 48, 200/48 = 4.17 microliters.
Thank you, Ivan :-) I just replied ElHO - one of my problems are the calculations. You can see my reply. I will aprreciate if you put some light in my chaos.
I try to perform two step RT-PCR on material from parafin embedded sections. So far I tested several primer pairs (one R and several F) amplifying different fragments - it worked fine with lower amounts of DNA. I have tested higher amount - no signal.
Yesterday I got signal on my gel, but it was for a product, whic does not span any introns - so, it means that the reaction works, but can not say if the amplified product is from cDNA or gDNA. The other pair, which spans intron did not amplified anything.
I just repeated the RT reaction - the cDNA values are very low. I started to think either the RNA isolation went wrong, or one of the F primers has stopped working. Is the second possible?