PCR no amplification - (Sep/03/2012 )
@ascacioc: As far as I know, the 100 ng of human gDNA in reaction is like golden standard. When I'm working with other templates I recalculate to equal number of target gene copies, because It's actually number of copies that matter (however I understand it's more complicated than this, since generaly more complex templates amplify differently). Usually using my web aplication for copy number.
But practically gDNA is not that sensitive to the exact amount in reaction. I usually don't even measure DNA before PCR and just put there 1 ul, it works It will work with 50 ng, 300 ng, 500 ng.. generaly not more than 1 ug. Since all isolation procedures and blood volumes we use are similar, the concentration doesn't vary that much, usualy it's around those 300 ng/ul. In that case I don't bother to dilute it for PCR and specific concenetration, because it doesn't matter. If there is extremely high concentration then I dilute, because reaction would fail.
Of course there are methods where the concentration should be adjusted, for any real-time quantitation generaly less than 100 ng is used, because it's very sensitive. Especially with SYBR assays the increased overal DNA concentration causes high background level fluorescence. For special aplications like HRM even less is used, between 5-30ng in reaction, all samples adjusted to the same concentration.
When you ask someone how much gDNA to put in reaction, he will usually tell you 1 ul as a joke. It's not a joke though
ascacioc on Tue Sep 4 12:30:38 2012 said:
we never know what the people asking here are actually using as a template
Yeh, that's what I meant, that we don't know so it could equally be plasmid or genomic template- but I was also having a bit of a joke with Trof and never meant my post to sound too serious
i am using At genomic DNA as a template and i have also tried with 3% dmso this time and annealing 58,60 62 64 66 68 and early denaturation 95 3 min and extension 72 for 1 min
my amplicon size is 1.5kb
At = arabidopsis thaliana? I know that the plant genome is a bit tricky because it is GC rich. Now, a lot of what you are stating makes sense. Indeed you need a modified protocol and still it will not work. I hope there is someone here with knowledge about it because in between the people who replied until now, I don't see smb with experience in plants genomics (I hope I don't offend anybody here, but Trof human genomic DNA as far as I understand, for me synthetic genes that come in little plasmids is the way...)
I have worked with human GC rich regions only, but 5% DMSO was the best fit for most, you can try up to 1 0%. It still amplified but didn't had such bright product. In my case 1 % was not enough, if you have more GC rich template, maybe 3 % is still not enough, try more concentrated.
What about the Primer3 Tm values? They usually work for me with Ta -4 degrees from this calculation. Tm's can vary a lot, that's why I'm asking about algorithm I'm comfortable with.
And another thing, did you checked the DNA is free of inhibitors? Do you have any working assay on the same species, so you can exclude potential problems with template?
I do most of my work at the other end, with organisms typically having 25% GC. But the few times I've had this problem, the magic ingredient has been betaine (Qiagen Q-solution). Add 3-8% of a 1 M solution (available prepared from Sigma, for just this purpose). Don't forget to use a longer extension time. And you never answered if your primers are just binding with 27/28 bp to your template, or if you have a short binding region and 5' RE sites.