At 05:56 PM 9/15/00 -0400, Hal Noer wrote:
>ABI recommends a 20ul sequencing reaction, however many people are running
>fractional reactions. Does anyone know how prevalent the 1/2 and 1/4
>reactions are?
>
>Thanks for your help- A lurker.
>
>1/2 and 1/4 sequencing reactions are probably far more prevalent than the
full version reactions, which tend to be used for difficult templates only;
This is especially true since the advent of big dyes which are at least 2x
as sensitive as the original rhodamine terminators.
4ul of big dyes in a total volume of 10ul should suffice for all high
quality templates of average G-C content below 20kb; Above that figure, e.g
when using BAC's for instance 8ul of big dye ( or more ) is normally
advocated.
In addition, some people find that using more primer helps :
When I ran a DNA sequencing facility in England, I normally recommended
using 5 p moles of primer ( ABI would advocate 1.6 for 1/2 reactions ), 4
ul of big dye and 50ng of DNA per KB of template plus insert for templates
upto 1KB - typically PCR products - and 100ng per KB of template plus
insert for anything above 1KB and smaller than very large templates such as
Cosmids and BAC's.
Moreover, (for PCR) products below 1KB and extension time of 2 min at 60
rather than 4 min at 60 will suffice; Infact, it may actually help since
the problem with smaller templates sequenced from clean preps tends to be
too much signal, which can distort the sequence, rather than too little,
which tends to be more of a problem when sequencing say plasmids.
Regarding 1/4 reactions, these should also work perfectly well in the
context of big dyes if the template is clean, i.e devoid of salt, average
G-C content and PUC derived vector; Some of the more elaborate expression
vectors tend not to work so well with smaller quantities of big dye, part
of the problem being ( I suspect ), secondary and/or higher order structure.
Also, PCR products below 1KB and especially below 500bp sequence better in
1/4 reactions for reasons stated above, i.e problems with too much signal
strength.
Finally, 2ul of big dye in a total of 10ul tend to favoured over 2ul in 5ul
totalon account of having more volume to play with; Also, using hot lids
and no mineral oil, residual evaporation in the context of 5ul reactions is
a significant problem.
Finally, for the 2 in 10 reactions still use 50ng per KB of template.
Hope this is of some use.
Best wishes,
Laurence S Hall
Laurence Hall,
Einstein Genome Centre,
1695 Poplar Street,
New York 10461.
Tel. (00) 1 718 405 8380
Fax. (00) 1 718 405 8383
E-Mail : LHall@megabase.aecom.yu.edu
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