POLYACYRILAMIDE - SEPARATION OF FOUR BASE PAIR DIFFERENCE (Aug/16/2010 )

Hi,

I need to seperate two DNA bands of size 236 bp and 240 bp. I used 12% polyacyrilamide gel(acrylamide: bis- acrylamide 29.0 : 1.0). The running buffer was 1XTBE and it was the same as the buffer in which the gel was cast. My PCR products were diluted to different concentrations before loading. The gel was run for 6 hours/ at 300 volt or it was run for 24 hours/ at 75 volt at the same conditions.(I used ice blocks)I used ethidium bromide for staining (100 ml 1XTBE / 10 ul of Ethidium Bromide Solution <10 mg/ml>). I stain the gel 15 min.

But eveytime I saw a smear and my bands couldn't be seperated or I couldn' t see them because of the smear.

I think I' m in trouble...Please help me...

You're in trouble. This will be very challenging with ordinary gel systems. Can you cut one or the other with an enzyme? Shorter fragments will be easier, even if both cut. There certainly are ways of doing this -- every sequencer separates out single base differences easily at this length. A Transgenomic Wave or other DNA HPLC system could do this. Mass spec could do this.