overcoming seq. bands in all 4 lanes

daj (David Johnston) daj at nhm.ic.ac.uk
Fri May 13 02:01:27 EST 1994


>Jonathan Hecht <jhecht at ucsd.edu> writes:
>
>>In article <jcn-060594092912 at 128.42.25.78> Jeff Nichols, jcn at rice.edu
>>writes:
>>>Does anyone have suggestions about overcoming bands in all 
>>>four lanes when using Sequenase?  I'm doing dsDNA sequencing.
>

try:
(A) doing the labelling step on ice for 3 mins
(B) add 10% DMSO to all steps as below

(1) Primer annealing

DNA (1.5pm) in H2O              - 6ul
primer (1.5pm) in H2O           - 1ul
DMSO                            - 1ul 
Buffer (5x)                     - 2ul

(2) Labelling
DNA/primer from (1)             - 10ul
0.1M DTT                        - 1ul
dGTP labelling mix              - 2ul
35S-dATP (600 Ci/mM)            - 0.5ul
DMSO                            - 0.61 ul
Sequenase                       - 2ul
(optional Manganese             - 1ul - to read closer to primer)
leave at room temp for 3 mins. If your template is a bit dirty and, despite 
the DMSO, prone to stops, do this step on ice for 3 mins (it does no harm 
whatsoever and I tend to do it routinely these days for all samples). You 
can premix the DTT, DMSO, labelling mix and 35S dATP for your days 
sequencing to minimise pipetting (I have not tried long term storage and I 
don't think you can premix the manganese (check in Sequenase mannual))

(3) Termination
labelled DNA from (2)            - 4 x 3.5 ul
ddG or A or T or CTP             - 2 ul
dGTP reaction set extension mix  - 0.5ul (extends sequence approx 2K bases) 
DMSO                             - 0.28ul
37C for 3 mins (have tubes with termination mix/extension mix/DMSO 
prewarmed before addition of labelled DNA)

(4) Stop
Stop soln                        - 4ul

store @ -20C

Hope this helps

Cheers
DAJ

David A. Johnston
Dept of Zoology, The Natural History Museum, Cromwell Road,
South Kensington, London SW7 5DB. England
(tel 071 9389297, fax 071 9388754, email daj at nhm.ic.ac.uk)



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