Difficult template

Laurence Hall lhall at megabase.aecom.yu.edu
Thu Mar 29 20:13:13 EST 2001


Dear Sabina,

the methods talked about thus far, viz. adding 5% - 10% DMSO, using dGTP or
mixing dGTP with Big dye should be used in conjunction with a more
aggressive cycling regime if you are not already doing this, i.e 

98 1 Minute...............x1

98 30 sec
(Tm -5) of primer 15sec
60 4 min...................................x30

The extra cycles compensate for poor cycling efficiency caused by tag being
to unable to navigate a high proportion of DNA molecules in consecutive
cycles.

The initial deaturing cycle - colloquially referred to as a 'Hot start' -
offsets this poor efficiency by maximising the number of single stranded
molecules available for sequencing

If you havn't done this already try customising your annealing temp. by
substituting the Tm -5C of your own primer for 50C, universally advocated

Also, whatever quantities of big dye you normally use for sequencing, try
doubling them to encourage product formation by the law of mass action and
to offset taq denatured by temp. of 98C in concert with chaotropic solvents
like DMSO ( the half life of taq decreases significantly above 96C and this
loss is exacerbated by DMSO, especially at conc. above 5% !!)


If the suggestions already mentioned still yield poor results, you may want
to think about the following :

1. Try using 0.5M betaine instead of DMSO : Preliminary data suggests the
latter can be more effective

2. Try titrating the conc. of Mg 2+ in your sequencing reaction from O.5mM
- 4mM ( Ordinarily, this conc. is 2mM final )

3. Design a primer close to the offending run of C's with a Tm of 70C and
try using  a two step cycle sequencing reaction in which annealing and
extension are combined, i.e 


98 1 min..................x1

98 30sec
65 4 Min..................x30

By designating the priming site close to the offending run of C's you
discourage intra strand binding and hence secondary structure

Moreover, if you use dGTP, because incorporation of dITP is circumvented -
in the context of big dye taq struggles to incorporate dITP efficiently at
temp. exceeding 60C -  you could design an even a 'hotter' primer and
elevate the annealing/extension temp. further; I have used this trick with
significant results !! Regardless though keep the annealing/extension temp
at Tm -5C

Finally, the above may even work with a 4:1 mix of Big dye : dGTP instead
of straight dGTP.

If anyone has any questions regarding the above, do not hesitate to contact
me.


Best wishes,


Laurence S. Hall   
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Laurence Hall,
Einstein Genome Center,
1695 Poplar Street,
New York 10461.

Tel. (00) 1 718 405 8380

Fax. (00) 1 718 405 8383

E-Mail : LHall at megabase.aecom.yu.edu


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