Annealed oligos vs. non annealed. Electrophoresis pattern.oligo annealing shrna
Posted 10 December 2012 - 10:47 AM
I'm cloning a shRNA sequence in plko.1 puro vector. I've already done the annealing step for the oligos, and now I'm trying to see whether they're annealed or not. I'm loading the same amount of either annealed or not annealed oligos and running a 0.8 agarose electrophoresis gel. My question is: Which oligos should run further? I'm getting a band at 25kb more or less for the non-annealed oligos, but I'm not able to see the annelaed ones.
Thanks a lot!
Posted 10 December 2012 - 12:12 PM
Single stranded should run a bit faster I think, but the difference will be very slight.
Posted 10 December 2012 - 02:29 PM
Posted 10 December 2012 - 06:44 PM
The easiest way to check if your oligos are annealed would be to do the ligation into a vector and then amplify across the insert site with sequencing primers.