discussion of DNA sequencing

Cheng Luo cheng.luo at plantphys.umu.se
Thu Jul 10 08:01:22 EST 1997


Of course, the RE sites in polylinker will be always there whatever you
cloned(specific one or any other shit ones) with the TA cloning vector.
You can not use REs which locate in vector to verify the clone it is
because both directions' primers located beyond the insert and
polylinkers. 
There are many reasons to scroll up the automatic sewuencing, but
cycling for sequencing  is a key step, the ready cocktail is rather
stable and reliabel, and usually the condition:96 10'', 50 5'', 60 4' 25
cycles in Perkin elmer suits to most of DNAs, then properly precipitate
the cycled DNA, and wash with 70% of ethanol.  And the pellet may not be
visible, but do exist, you can not wove away by wash or vacum dryer.
of course you may need to tell much more, for instance the Sp6 primer,
it is extremely high ambiguities? what kind of service system you are
using, and so on. 

Wish this help!  

Cheng



To: methods at net.bio.net

From: ddsyr at ns.east.cn.net (ma)

Subject: Discussion of DNA Sequence

Date: 10 Jul 1997 01:28:08 -0700


Dear Netters:

I have met many problems in DNA sequence recently, so I like to initiate
a discuss on this topics. I cloned a PCR product and insert into a
T-vector. The enzyme check of polylinker is OK. The sequence from one
end (T7) by DNA automator is OK. But the sequence from another end
(SP6)did not get any signal. The sequence was conducted by a commercial
company since our lab has no such equipment. Could any one give your
idea what is wrong about it? I also like to know the general
troublesomes in DNA automation sequence and how to avoid them? Any
seggestion and discussion is greatly welcome.



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