Automated DNA Sequencing - better gel composition

Sudarshan Sharma ruhi at BIOCHEM.IISC.ERNET.IN
Fri Aug 9 09:38:09 EST 1996


Hi Friends 
	My name is Sudershan Sharma and I have been working with 
automated DNA sequencer (ABI PRISM 377) for the past one year. I get 
better resolution with following gel composition:

	23:1 Acrylamide: bis-acrylamide 30%   : 7.5 ml
	18 g of Urea (6 M) 
	6.0 ml 10 X TBE
	22 ml MilliQ water
	
	Degassing time 8 minutes

	10% ammonium persulphate :300 microliter
   &    TEMED : 23 microliters

	This resolves 550 bases with 99% accuracy and less than 1% Ns
when run at 2600 V, 150 watts for 4 hours. The gel cast was 36 cm long 
with shark tooth comb. 

	For a 2X run following conditions also gave improved resolution:

	23:1 Acrylamide : bis-acrylamide 30% : 6.5 ml
	6.0 ml 10X TBE
	21.0 g Urea (7 M)
	make up the solution to 50 ml with MilliQ water
	Polymerise with 300 ul APS and 23 ul TEMED
	run at 1580 V, 50 watts 8 hours.

	This resolves 650 - 680 bases with 99% accuracy and less than 1% 
Ns. The gel cast was 36 cm long.

	Hope this sees a few people out of there troubles.

							Bye

                                                   Sudershan Sharma





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