Cycle Sequencing conditions

[Holger Melzl]

Dear Barbara,

For urgent Samples we use the following conditions:

96°C - 10 s
50°C - 5 s
60°C - 2 min for 25 - 30 cycles

this gives a total cycling time of 1.5 - 2 hrs (depending on
your cycler)

10 µl reaction w/ 4 µl BigDye Kit (sometimes 12 µl reaction
/ 4 µl Kit)

We use about twice the amount of template compared to
regular sequencing
reactions and the same amount of primer as in regular
reactions (6 pmole).

Maybe even 15 cycles is enough if you add even more template
(but doesn't give optimal sequence quality). Sequence
quality seems to decrease a bit with fewer cycles (more background).

We've used these conditions with PCR-Products of ~300 bp,
but it seems to 
be possible to get greater read length (400 or 500 bp), too.
Though signal
strength will decrease towards the end. 

ABI Germany sometime ago recommended a two temperature
cycling protocol with:

15 cycles of
96 °C for 10s
60 °C for 90s

using either:

1.5 µg of plasmid DNA, 10 pmole primer, 8 µl BigDye Kit in
20 µl
or
200 ng PCR product, 10 pmole primer, 4 µl BigDye Kit in 20 µl

which should cut down cycling time to 40 min. (as proposed
by ABI)

I've tried this only once with a PCR-Product, but sequence
quality didn't 
seem to get as good as with a 3 temp. cycling protocol.

Maybe there are other (better) ways to decrease cycling time.
I was wondering if it was possible to use for example
Boehringer Mannheim's LightCycler for sequencing reactions
(should do 30 regular PCR-cycles in about 10 min).


Hope this helps
CIAO

Holger


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 With compliments...           
                                               
                 Holger Melzl 
             DNA-SEQUENCING SERVICE          
   Institute for Medical Microbiology and Hygiene
          University of  Regensburg   
               
         Franz-Josef-Strauss-Allee 11         
               D-93053 Regensburg
                    GERMANY              
      
    dna.sequencing at klinik.uni-regensburg.de
      holger.melzl at klinik.uni-regensburg.de                 
           
           
           Phone : +49 (941) 944 6474          
           Fax   : +49 (941) 944 6402  
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