PCR: Band in one lane, streaking in the other..HELP!
Posted 08 July 2011 - 07:47 AM
We are doing a PCR (1.2KB amplicon)with genomic mouse DNA and a vector to simulate a positive genotyping PCR. Vector is diluted so there is one copy per genome. 50ng of genomic mouse DNA are used.
Our problem is that we get streaking in some lanes, and the specific band in others. Picture attached.
What we have tried:
-New genomic DNA
-New diluted vector
-Fresh TAE to run the gel
-Fresh SYBR gold to stain the gel
-Using a different thermocycler
In the picture, everything is from the same master mix... so we really don't know what to think.
Any help is appreciated!
gel024.pdf 98.67KB 313 downloads
Posted 08 July 2011 - 09:33 AM
..."best of our knowledge, as far as we know this had never been reported before, though I can't possible read all the published journals on earth, but by perform thorough search in google, the keywords did not match any documents"...
"what doesn't kill you, makes you stronger"---Goddess Casandra reminds me to be strong
"It's all just DNA. Do it."---phage434
Posted 12 July 2011 - 07:00 AM
Try do a gradient dilution of your genomic DNA. I think your genomic DNA is way far too much...
Second what Adrian has said.
Too much DNA.
Thanks for the response guys.
We have tried a control with just the vector (no genomic),and we still get the hit or miss streaking. We will try less genomic also, but we are know thinking it could be hyper coiling of the vector. We are liniarizing that.
What do you think? Any other ideas?
Thanks a bunch!