need help for the Cy5 sequencing reaction

bo pang pangbo at chem.bu.edu
Thu Jun 15 13:19:58 EST 2000


I am using the Cy5 Dye primer kit to sequence my plasmid DNA, however i
can't get a good reproducible results. That is the protocol for my
sequencing:
1) Aliquot 3ul each of the A,C,G,T termination mixes into their labeling
tubes respectively
2) Using the enzyme dilution buffer supplied in the kit, dillute the
necessary quantity of the Thermo Sequenase enzyme 1/10 in a seperate tube.
mix, microfuge and place on ice. 
3)make a master mix:
          sequencing buffer                         2 ul
          Cy labeled primer (3 uM)                 1.0 ul
          Plasmid DNA (250 ng/ul)                  2.0 ul
          dd H2O                                   5.5 ul
         diluted Thermo Sequenase                  2.5 ul
          Final volume                             13 ul
4) Aliquot 3 ul of the master mix into each of the 4 termination tubes,
Pipet gently up an ddown during each steps.
5) Place tubes into a preheated thermo cycler and start cycling programn.
6) cycle sequencing program:

         Denaturation      94 degree   3 min    cycle 1x
                           94          15 sec 
        first stage        55          15 sec   cycle 18x
                           70          60 sec 
                           94          15 sec
        second stage       70          60 sec   cycle 15x
        soaking            4          forever
7) load the sequencing samples into the gel

Actually this protocol is for the single strand control DNA, and I use it
to sequence my double strand plasmid. It seems that this approach works on
the control ok, half of time i can get the  decent signals. however when i
sequence the plasmid, mostly i cound only get messy data. Every time i
used the same procedure, and I am pretty sure that I didn't
technical mistake in the experiment. I am wondering why i can't consistant
data in the sequencing reaction. Do you have suggestion for my sequencing
protocol? Thank you very much in advance.

Bo






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