gDNA on a gel? - (Aug/03/2005 )
What is the minimum ug/ng amount of gDNA (Genomic) necessary to visualize on a agarose gel?
I'm not sure but I think that with 200ng you can see your gDNA.
If the DNA is uncut, and sticks in the well, you should be able to see < 10 ng of DNA. If you are cutting it and visualizing the bands, each band will need to be about 10 ng or so to be visible, so you need much much more total DNA -- a few micrograms, e.g., since you will have hundreds or thousands of bands. I assume you are staining with EtBr or SybrSafe.
Ok, so I am trying to amplify two regions, ~600 & 1250 bp long from genomic DNA but I am having issues optimizing my PCR conditions.
I have tried various annealing temperatures in 1 degree increments from 50-58 C (my primer TMs are 60.12 and 60.19 respectively designed through primer 3) Amplification <53C results in no bands and between 53-58, results in no product band but in a ~50bp band which I believe to be a primer dimer. My only option at this point seems to be to run a magnesium titration from .5-4 mM at each annealing temp to see if I can get my product to appear or to push my annealing temp up past the primer Tm. Can anyone recommend anything else I could try? Is it ever likely that the annealing temp for primers is above the calculated Tm?
Are those primers degenerate or specific? if they are degenerate I suggest making the primers again with different restriction sites tagged on their ends to improve amplification, although those high TMs suggests that you may have to shrink them a bit as well
How much dna are you using in the PCR? the optimal concentration of template in a PCR if you're using gDNA is 1-10ug/mL which works out to be 0.5ug for a 50uL PCR reaction (max)
Did you extract the DNA using a kit? kits usually use filter columns which are a hell of alot better than any protocol without one. Any DNA extraction procedure without one brings to my mind the term "bucket chemistry" you may want to check the purity (A260/A230 & A260/A280) and concentration (A260) of your gDNA.
Hope that helps
The DNA was isolated via gravity flow columns in qiagens RNA/DNA kit, 260/280 ratio was 1.79 or therabouts with about 22 ug of DNA being isolated from a T-75 flask of cells. I was originally running about .7 ug of genomic DNA but a colleague recommended that I drop that down to less than 100 ng. So I am trying a magnesium/annealing temp gradient on 50 ng of gDNA template.
The primers themselves are designed to specifically amplify my region of interest. Ultimately this DNA is going to be carried over to bisulfite treatment for epigenetic methylation study. But I wanted to be sure that I was capable of amplifying my non-converted template before I continued.
My MM for the PCR consists of:
200 uM dNTPS
1x Taq Buffer
MgSO4 conc ranging from 2-4 mM
.5 uM F + R primers
2U NEB taq pol
dH2O to 50 microliters
When genomic DNA is run on a gel should it appear as a streak or a single high molecular weight band near the top of the gel?
I don't usually work with genomic DNA but I ran some genomic DNA that I isolated from the qiagen rna/dna kit, along with the same DNA that had been restriction digested with HindIII on a low percentage agarose gel. The intact gDNA appeared as a single strong band near the top of my gel (30-40 kb or higher going by the ladder) with a small amount of smearing and with the restriction digested sample, the band dissappeared and strong smearing appeared. Is that right? Or should the restriction digest resolve the gDNA into at least some visible bands?
That sounds completely normal for a genomic DNA digest.