IUBio

Cycle Sequencing of cosmid templates?

Bill Bill
Tue Jul 1 08:20:25 EST 1997


     Hi Armin,
              Have you tried the TR Mutation enzymes such as the 
     Thermosequenase. This is a very robust enzyme and the following 
     protocol works well in my hands:
              Template         8 ul (About 5 ug of Cosmid)
              Primer           4 ul (4 pmoles of labelled primer) 
              Reaction buffer  1.6 ul
              T.Sequenase      4 ul (8 Units)
     
                   Split the above into 4 X 4.4 ul and add to 1.6 ul of 
dd 
     Termination mix. The termination mixes to try depend on the read 
length 
     needed and the Fidelity of sequence. I would try the following 
Ratio's - 
     these work well in my hands:
          1. 3:300 uM ddNTP:dNTP
          2. 6:600 uM ddNTP:dNTP
          3. 5:500 uM ddNTP:dNTP
          4. 2.5:750 uM ddNTP:dNTP
     
      Cycle conditions:
     I would try to avoid low annealing temperatures - the Tm of the 
primer is 
     very important, taking into consideration the Salt conc. and Primer 
conc. 
     Due to the length of the template, use a primer that is more 
specific than 
     normal - ie longer ( 24 bp or so). Try to use equimolar amounts of 
     primer/template. The shorter reading sequencing mixes will be ideal 
for 
     accurate sequence cosmid walking. An alternative is the Sequitherm 
enzyme 
     (I don't have the protocol at the moment as I am travelling in Asia) 
- a 
     good workhorse. You can also add DMSO to about 5%, and Tween - 20 to 
about 
     1% for denaturation purposes.
               95C   2 mins.
                  94C        45 secs.
                  Tm + 10C   30 secs (ie try 60C)
                  72C        45 secs
                          X    25 cycles
                          Add 4 ul Stop (Dextran Blue at 6 Mg / ml in 
formamide)
     I should end by saying that these protocols are developed for The 
ALF 
     Express Single Dye system. If you use the high DNA conc. on the ABI, 
a 
     precipitated pellet may cause running problems in the gel.  Also, 
the 
     various nucleotide mixes will not be relevant to Dye labelled 
Terminators.
                          
        Bill Vallins
     
______________________________ Reply Separator 
_________________________________
Subject: Cycle Sequencing of cosmid templates?
Author:  Armin.Michel at net.bio.net at Internet-Europe 
Date:    24/06/97 06:17
     
     
Does anybody know of a reliable protocol for sequencing cosmids by cycle 
sequencing? Of special interest for me are the amounts of DNA template 
and primer oligonucleotid to be used in the cycling reaction. 
I tried the sequencing reaction with standard template amounts for 
plasmid DNA (about 500 ng which yielded good results in our standard 
plasmid sequencing reactions) and with significant higher template 
concentration (about 2 ug) but got no usable sequence using standard 
sequencing protocols referring to the ABI 373/PE ABI Prism Cycle 
Sequencing manual. 
In addition, I am also looking for a cosmid DNA purification method 
which yields good amounts of (sequencing-grade) clean DNA. Up to now, I 
used QIAGEN column purification but this method resulted in very low 
yields.
     
Every help is greatly appreciated.
     
Thank you in advance,
Armin Michel.
     
Institute of Human Genetics
University of Saarland
Germany



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