ds sequencing troubles

[Brian Foley]

jo_c at vcp1.vcp.monash.edu.au wrote:
: I have been having trouble with ds sequencing ...
: My sequencing gels either give me nothing or bands in all lanes.
: I've tried using a control without my insert and I get faint
: bands but they seem to be in all the right places. 

	I have had very good results sequencing ds-DNA if the template
is prepared by either alkaline lysis/CsCl gradient (milligram quantity 
preps) or alkaline lysis/glassmilk (mini-preps 20-30 micrograms of DNA).  
And very poor results, or highly variable results if the template was 
prepared by alkaline lysis/phenol extractions.

	The glassmilk procedure is in the bionet.methods FAQ.  It does 
not always give a high enough yeild of DNA (in my hands) but I can tell
from a quick agarose gel if I will be able to sequence a prep or not.
If there is enough DNA, it always sequences beautifully.

	With other mini-prep procedures (I've tried quite a few), I
sometimes get great results and I sometimes get unreadable gels.  A
major waste of time and source of great frustration!  I still haven't
nailed down the variability in yeild of my glassmilk preps, but I 
get around it by growing a couple 3 ml cultures of each clone and 
harvesting them both.  Often one of the two side-by-side preps gives a
better yeild than the other.


--
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*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *
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