I have been doing click chemistry reactions with DBCO TEG 5' modified DNA and RNA modified at the 3' end with Azidohexanoic acid. I also use a splint of 30 bp with HEG in between the 2 homology arms.
Previously had good results with an obvious product of the reaction in my PAGE but recently I have changed the sequence of the components and it's not as good.
The PAGE has been 15% Acrylamide 8M Urea in 1x TBE with 2x RNA loading dye from NEB. I heat denature for 10 minutes at 80 degrees. I also prerun for about 1 hour at 70-90V in 1x TBE then rinse the wells before loading. I run it for at least an hour.
The size of the RNA is 100 nt and the DNA in the first set of experiments was 90 nt it is now 127 nt, the splint is the same size. Can anyone advise whether I need to change the way I have been doing the PAGE or if there is something wrong with the reaction?
1st image is very bad because I forgot to rinse the wells and there are holes in the gel.
2nd image is the same as first except 15% instead of 12%.
3rd image is what I was getting previously with the old oligos.
I need to get the first two looking the 3rd image basically, any ideas?
I would not prerun more than 30 min, it may produce heat which is responsible for deforming the DNA or RNA band.I also clean the well by rinsing before the prerun as well. Sometime if my gel image looks bad, I prepare fresh TBE , acrylamide soln. I check the quality of DNA and RNA to make sure everything right. It helps me to know which component went bad.
Thanks Rabiul, I will try and do it again and this time pre-run it for less time. Thanks very much,
Dan.