template preparation for cycle sequencing

"Alexander Kraev" bckraev at aeolus.ethz.ch bckraev at wawona
Fri Feb 24 03:58:51 EST 1995


In article <Pine.3.07.9502231130.A6330-b100000 at wagsun>, marchaa at agric.nsw.gov.au (A.Marchant) writes:
>I have been trying to sequence PCR products using two Promega kits, the
>'Silver Sequence' system, and the 'f-mol' system.  Both of these use
>cycles of annealing and extension, and strand separation, and are done in
>a PCR machine.  I started with the silver sequence system, and got signal
>too low to be readable.  I tried upping the template concentration, and
>could see some faint sequence bands (not readable) in the +ve control (their
>supplied plasmid DNA).
>
>I then switched to their f-mol system, which is exactly the same, except
>you end-label the sequencing primer with 32P.  This should have given me
>much more signal, and so give me a better idea of what's going wrong (my
>intention is to go back to silver staining when it is all debugged).
>
The fmol sequencing mixes are not exactly the same as those in Silver Sequence:
the latter are kind of a "long read" version of the former. Besides that, the
Silver Sequence protocol makes you use much higher amounts of DNA. You will
get great results if you use the Silver Sequencing protocol and label primers
with 33P! However, I think you should first try to get the control reaction
working. If you can't, there may be a problem with your hardware: e.g. the
cycling is too slow. Also note that the control primer is 24 base long! Your
old primers may perform well or not anneal at all at 70 C! Good luck


*************************************************************************
Alexander Kraev, Ph.D.                 Internet: bckraev at aeolus.ethz.ch
Lab. of Biochemistry III               Phone: 0041-1-632-31-47
Swiss Federal Institute of Technology  FAX:   0041-1-632-12-13
Universitaetstr.16
CH-8092 Zurich
"Some ideas are obscure not because they are complex, but because they 
 are excluded from our circle of comprehension" - Kozma Prutkov



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