At 04:44 PM 9/29/00 -0700, Gabriel Archdeacon wrote:
>hello all,
> i am hoping some of you might be able to help me. i am trying to run
>sequencing reactions on a double stranded plasmid template. the problem is
>that the plasmid is 56 kb. i have a protocol that has been successful for
>plasmids up to twenty or twenty-five kb, but nothing for a plasmid this
>large.
>
> i ran some reactions today as follows:
>
>template 2ug
>primer 16pmol
>
> i used a reaction volume of 40ul. the cycle sequencing conditions were
>as follows:
>
> 95 for 5 minutes
>
>
> forty cycles of
>
> 95 30 seconds
> 50 20 seconds
> 60 4 minutes
>
>
>followed by a 4 degree hold.
>
>these conditions gave no results.
>
>
>If anyone thinks they can help, or has any suggestions please let me know.
>I thank you all in advance,
>
>--
>gabriel archdeacon
>elim biopharmaceuticals
>
>gabriel@elimbiopharm.com
>(650)-827-1984
>fax (650)-827-1986
>
>
> Dear Gabriel,
sequencing something this large short of what you are already doing is
going to be tough; Conditions advocated for something as large as 25kb are
likely to be the same modifications as those advocated for something like
50kb.
If those conditions havn't worked thus far, also try the following :
1. Linearise your plasmid: Secondary/tertiary structure is a major
consideration for something this large and linearisation will eliminate the
latter; Traditionally, large templates such as cosmids were linearised
prior to sequencing for these reasons.
2. Add 5 % DMSO and increase your denaturation temperature to 98. This will
deal effectively with secondary structure although be aware that you may
lose some taq by combining 98 with DMSO. However, advocating one or the
other - the traditional way to deal with GC rich template of small size (
below 10kb ) - is probably not sufficient for something this large if
secondary structure is an issue - do you know if the GC content exceeds 60
% ? - and in any case if YOU USE MORE THAN 8UL OF BIG DYE, YOUR RESERVES
SHOULD BE ENOUGH.
3. If the above fails, try repurifying your template with PEG-NaCl,
requantifying then repeating the above ( Talk to me if you wish about PEG
ppt. )
4. If that fails, try sequencing your plasmid with dGTP chemistry rather
than big dye : Incorporation of inosine is rate limiting and sometimes if
the inosine load is high, such as in the case of a GC rich template or if
the plasmid is large and therefore absolute incorporation of inosine is
also high taq will struggle with signal strengths. In these situations in
my experience dGTP will lift the signal strength sufficiently to give
readable sequence whereas the same template sequenced with ordinary big dye
can be noisy.
5. If that doesn't work - Titrate the Mg in the sequencing reaction : The
final working conc of Mg is 2mM; Try increasing to 3mM and also decreasing
to 1 mM.
6. If that doesn't work - Try a combined annealing/extension temperature of
55C. This can work with primers that are ordinarily used at 50C but some
signal strength might be lost. However, you are using 40 cycles and the
higher stringency of 55 should ensure that background noise isn't a problem.
7. If that doesn't work - Placating the Gods through some sort of sacrifice
might be in order !!
Hope some of this helps,
Laurence
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********
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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