Separation of RAPD fragments on agarose

Mnr AA Myburg Plant & Dierkunde x2818 paam at rs.uovs.ac.za
Thu Jun 29 06:59:43 EST 1995


Anybody doing RAPDs out there,

I am experiencing problems separating my RAPDs on agarose. I am screening 
wheat DNA for resistance markers using AB taq and I am running the samples 
in 2% agarose on the Hoefer HE100 Supersub. I am separating my samples at 
5 V/cm (150V/30cm for 3h - 15 cm gels)

When I use 1XTAE buffer, the bands (especially those larger than 1 kb) are 
fuzzy and distorted, } - shaped. 

The HE 100 manual suggests the use of TBE instead of TAE because of the 
lower buffer capacity of TAE. I have tried TBE buffer, but all the 
bands larger than 1 kb are smiling, ) - shaped. The lower bands are perfect.
I have changed the loading buffer from a glycerol base to 15 % Ficoll 
BFB. This has improved the seperation, but only for the lower bands.

I have also tried lowering the agarose concentration to 1.6% and running the 
gel at lower V (3 V/cm), but the >1 kb bands are still smiling and smeared. 
I have also lowered my sample concentration to avoid overloading.

I would appreciate any suggestions which you might have. I still have to 
screen 3 segregating populations (100 plants each) and I am desperate!


Zander Myburg
paam at rs.uovs.ac.za




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