quick n' easy plasmid sequencing

rybicki at uctvax.uct.ac.za rybicki at uctvax.uct.ac.za
Thu Nov 21 05:53:45 EST 1991


In article <201153.292a4f30 at uctvax.uct.ac.za>, rybicki at uctvax.uct.ac.za writes:
> I would like to share a quick n'easy plasmid sequencing protocol we have
> recently lifted, which works really well.
> 
> Ku-chuan Hsiao : A fast ands simple procedure for sequencing double stranded
> DNA with Sequenase .  NAR 19:2787
> 
> Basically you simply dissolve clean miniprep plasmid DNA in 25-50ul TE;
> 
> to 5ul add 1ul appropriately diluted sequencing primer (10ng/ul);
> 
> add 1ul 1M NaOH & mix;
> 
> 37 deg C 10'
> 
> add 1ul 1M HCL & mix;
> 
> add 2ul 5X Sequenase buffer
> 
> (optional: 37 deg C 5 min)
> 
> and thereafter go for it according to the Sequenase manual.
> 
> Cuts plasmid sequencing time down drastically, and will really be a help when
> sequencing more than one template.
> 
> Ed Rybicki


Modification by Di James (Microbiology Dept, UCT)

FOR GC-RICH TEMPLATES

after addition NaOH, INCUBATE 40 DEG C 30 MIN

KEEP AT 40 WHILE ADDING HCl AND 5X RXN BUFFER

COOL VIAL TO ROOM TEMP SLOWLY!! (+/- 30 MIN)

Suggestion: allow tupperware with water at 40degC to cool on bench (with vial
in boat).


Good Hunting!!
dijames at micro.uct.ac.za



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