Automated DNA Sequencing - better gel composition
ruhi at BIOCHEM.IISC.ERNET.IN
Fri Aug 9 09:38:09 EST 1996
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.
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