Posted 07 June 2011 - 05:00 AM
I've been running a few different PCRs using plasmid DNA. When run on a gel, the bands are present (that is not the issue), but clarity is poor and all bands are a bit 'blobby'. For now, I am just testing ladders and this is not an issue. However, I want to use this PCR set up to be able to differentiate between fragments that are just a few bp apart - clearly if my bands continue to look like this, I won't be able to do so. Because the ladder looks the same as the bands, the issue is with running the gel rather than with the PCR itself.
I have used 1.5% and 2.0% agarose with TBE buffer with the same results. I have ranged the voltage from 60-80V and I have ensured that the buffer reservoir is full in all instances. I am not sure what is the next step I could take to resolve this issue.
Help would be appreciated,
Posted 07 June 2011 - 07:37 AM
I took a ruler, I measured you "well" height and length. I measure the same with your "band"... I find it is the same...
In other words, is your "well size" that makes it so. you try to use a well size of "10mm x 1mm" (or 0.8mm), your resolution will be improved.
..."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 08 June 2011 - 04:44 AM