Sequencing Gels

["Alexander Kraev" bckraev at aeolus.ethz.ch]

In article <2F60092A at SmtpOut.em.cdc.gov>, tar9 at NIOBBS1.EM.CDC.GOV ("Reid, Thomas M.") writes:
>
>Hi-
>I am trying to sequence an 800 bp cDNA amplified by PCR.  I am using 
>end-labeled primers and Sequenase (ver 2.0) enzyme.  I have 2 problems that 
>I would appreciate any comment on.
>
>I am getting strong termination bands approximately 250 bases away from the 
>primer.  This happens with all three primers I am using so it is not 
>sequence-specific.  My termination reactions contain a 20:1 dNTP to ddNTP 
>ratio.  Is there any way to increase the length of DNA that is polymerized 
>by Sequenase?

If you are using the kit, use the so-called extension mix, otherwise dilute
your sequencing mixes with 100 uM all four dNTP 1:1, 2:1 etc until you get
the desired range of extension products ( usually 1:1 is enough ). However,
regarding your second problem, you may achieve the same by using less template
( see below )
>
>  I am getting fairly good resolution of bands near the bottom 
>of gels and especially near the top of my gels.  However in the middle of 
>some (but not all) gels the bands are smeared quite a bit and are almost 
>impossible to read.
 
If your cassette is OK, then you most likely use too much template, the
smearing is due to excess contaminating low MW RNA ( gel overload ). Optimal 
amount is 2-3 ug, unless your plasmid is very large. 


*************************************************************************
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
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