To DNA sequencing experts!

Michael Cooley szcooley at dale.ucdavis.edu
Mon Dec 19 20:03:01 EST 1994


Mic Chaudoir (mic at nwu.edu) wrote:
: In article <1994Dec10.030247.20311 at news.snu.ac.kr>,
: jongslee at cd4680.snu.ac.kr (JongSub Lee) wrote:

: >  Hi experts
: > 
: > I wanna ask you something on sequencing.
: > 1) When my co-worker sequenced the same DNA sample, his x-ray film did not
: > show any bands at all sometimes and did show good bands sometimes.
: > He did sequencing with exactly same DNA sample aliquots.
: > Why did the results?

: My guess is technique.  The dsDNA sequencing techniques are not trivial,
: and inconsistent results are not uncommon.


: > 2) On the sequenced bands, there are some ladders on all 4 lanes. There
: > were no so many ladders but some ladders on the film. We cannot read the
: > sequence around the ladders. How can I avoid that? 
: > Is the sample quality problem? or primer-annealing problem?

: I am not sure what you mean, exactly ?  If you mean that you simply had a
: latter of bands in the lane, well, this is commonly seen as well. My guess
: is that it would be caused by bad technique and/or not-so-clean DNA

: Here are tips that we use for good results:

: 1)  Always use Cesium Pure DNA.  This gives the most consistently good results.
: 2) Make sure that all of your reagents are fresh
: 3) Do not over-extend.  Watch your times carefully.
: 4)  Pipette all mixtures with great care, avoiding bubbles


: Hope this helps.  A better description of the second problem would be
: helpful, though.

: -- 
: mic
: mic at nwu.edu
:  
: "It [PowerPC Mac] won't have any effect at all on Intel machines.
: They will continue to plod along, running the same clunky Windows              and wretched DOS drivel they always have.  The PPC will affect Intel PCs the same way an SR-71 Blackbird affects a dairy cow."
:                      Robert Rhode, in comp.sys.mac.advocacy


I had a similar problem a while back (referring to the sequence vs smear 
from the same DNA prep). The problem did in fact turn out to due to DNA 
purity and homogeneity. I could correct the problem, sort of, by 
pre-heating the DNA to be sequenced to 65 oC for 15 minutes before taking 
an aliquot. By sort of I mean that the sequence was uniform from sample 
to sample but poor quality, ie bands in all lanes some of the time. But 
you don't need to CsCl purify the plasmid. Just purify the alkaline prep 
with GeneClean or Wizard, ie any Kit which binds the plasmid out of 
solution and washes with a high salt solution. The above mention of 
watching carefully the extention times is also important.


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