Longest dideoxy sequencing read?

daj (David Johnston) daj at nhm.ic.ac.uk
Tue May 3 04:18:33 EST 1994


Hi, 

This is the modified sequenase V2 protocol which we use routinely and has 
read some 750 bases from primer with Hydrolink long ranger gels (we didn't 
try any further though I am sure we could have)

(1) Primer annealing

DNA (1.5pm) in H2O              - 6ul (CsCl or Quiagen purified plasmid)
primer (1.5pm) in H2O           - 1ul
DMSO                            - 1ul 
Buffer (5x)                     - 2ul
proceed exactly as per sequenase protocol (65C for 2 mins, cool over 30-45 
mins to 30C, then store on ice and use within 2 hours). 

(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


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)

(for long reads, use the labelling mix @ 2/5 dilution instead of the 
standard 1/5 dilution and double the amount of 35S. For mega long reads 
use @ 3/5 and treble the 35S etc (though I haven't needed to do this))

(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

Run on long ranger gel as per supplied protocols (5% gel run @40Cish) 
and you should get sequence labelled extending to several Kb. If you can, 
use 0.2mm gels (arghhhhhh!!!) and try to load the sample (2.5-3ul) onto the 
bottom of the well using an ultrafine tip, rather than let it fall through 
the well. We tend to time our gels wrt the bromophenol blue in the loading 
mix. When it hits the end, load some more stop solution in a spare track. 4 
lots of BPB to the end (or longer) should give you the sequence you want 
(with the long ranger, these upper bands will be pretty closely spaced but 
hopefully nice and sharp). It might be worthwhile trying 4.5% or 4% gels 
but I haven't done this myself.

Cheers and goodluck
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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