Posted 24 January 2012 - 02:08 AM
Currently, i'm trying to do some southern to check the number of copy of my transgene. I'm working on wine. After having loads of trouble to get rid of polysacchardides and polyphenol during dna extraction, i'm struggling now with the southern.
I'm working in a small lab and we don't have a lot of facilities and in this case we don't have a stratalinker to crosslink dna on the blot... Thus, i'm trying to do the crosslink with our geldoc. Do you think it's possible or the geldoc is not powerfull enough, because i've got no signal after revealing my southerns...
My other question is, how to force all the digested dna to run in the agarose gel. Because there's a lot of dna remaining in the wells after running...
Thanks for your help
Posted 24 January 2012 - 05:27 AM
You probably want to work your detection from the end towards the beginning. Spot serial dilutions of your DNA or a control on a membrane, and practice your detection. If you are using a probe, start by spotting serial dilutions of the probe, and detecting it. Then, serial dilutions of your sample, detecting with your probe. Only when that is working, start doing gels and gel transfers.
Long DNA fragments don't move from the well, so likely your DNA is not completely cut. This is probably not a significant problem.
Posted 24 January 2012 - 07:29 AM
Posted 31 January 2012 - 05:53 AM
Posted 31 January 2012 - 06:44 AM
Edited by David C H, 31 January 2012 - 06:52 AM.
Posted 31 January 2012 - 06:49 AM
Posted 31 January 2012 - 06:52 AM