Two things that have helped me are: TdT in my rxns and constant temperature
gels.
1) TdT helps more with false stops than compressions, but it REALLY
cleans up the rxns. I use it exactly as described in the USB COMMENTS
article "An Efficient and Reliable Method for the Elimination of
"Artifact Banding" When Sequencing Double-Stranded DNA Templates" Timothy
W. Fawcett and Sue G. Bartlett. I am sorry I don't have the date or
volume # but please write me if you can't find it an I will find a way to
send you my photocopy and/or my protocol.
2) In my opinion, constant temperature gels are CRUTIAL to the
elimination of compressions in GC rich Chlamy! Our lab purchased a
constant temperature power source from Stratagene. (You can also buy
them from Bio-Rad). If you can't go so far as to spend money right now,
try running your gels at higher power and monitor the temperature of the
glass plates to try to keep temperature close to 55 degrees celsius.
Please let me know if you need more info.
Julie
jknott at biosci.cbs.umn.edu
P.S. I do double stranded sequencing of Chlamy from both CsCl purified
DNA and Stet prepped DNA.
On 2 Jun 1995, Emma Berta Gutierrez wrote:
> Do you know the tricks to sequence Chlamydomonas DNA? I have lots of
> compresions in my gels.
> Thanks for your help.
> Ember.
>>
