Shortcut for Sequencing Kits

Gregory A. Denomme 37_527 at uwovax.uwo.ca
Thu Dec 19 15:59:09 EST 1991


     For those of you who have the Pharmacia T7 Polymerase sequencing kit
and are unable to take advantage of the method published by Ku-chuan Hsiao
(Nucleic Acids Research 1991;19:2787) since it relies on the Sequenase Kit
(USB), I have modified it to work with the Pharmacia Kit (no affiliation
with the company whatsoever).

***
     Dissolve the DNA in TE to achieve a concentration of 1.5 - 2.0 ug/4 uL
     (If higher conc. then dilute with sterile dddH2O)
     Add 2 uL of the sequencing primer
     Add 1 uL of 1N NaOH, mix, vortex (quick spin)
     Incubate at 37C for 10 minutes
     Add 1 uL of 1N HCl
     Add 4 uL of sterile dddH2O
     Add 2 uL of annealing buffer, mix ,vortex (quick spin)
     Incubate at 37C for 20 minutes
     ---continue as outlined in the Kit manual
***
Remember, this is for double stranded DNA sequencing and I find that any 
problems are due to inaccurate NaOH or HCl concentrations.

Good Luck
Greg



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