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Sequencing using thermocyclers other than the PE9600

Gys de Jongh GysdeJongh at csi.com
Thu May 13 16:53:56 EST 1999


Apart from the timing the MJ research machine has a much faster temperature
ramping. The manual lists a maximum of 3degrees/sec . The ABI Prism® BigDye
Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq® DNA
Polymerase, FS ;     Kit protocoll 4303237 Rev. C , page 16 says : If you
use a thermal cycler not manufactured by PE Biosystems, you
may need to optimize thermal cycling conditions, including ramping
rates. Contact PCR Technical Support (see page 10) for questions
about thermal cycling.

The protocol and a lot of other usefull information can be found on the
perkin elmer web page : http://www2.perkin-elmer.com/ab/  under :
services -> technical support  or directly at :
http://www2.perkin-elmer.com/ab/techsupp/gaprotocols.html

We use the pcr program recommended by perkin elmer ( on page 17 , see above)
with lower than the default (MJ Research) temperature ramping. For the past
two years this has given rock stable results. Here it is:

MJ Research PTC-200
20 microliter tubes
Use heated lid
Control method calculated

  1 =   0.9d/s to 96.0d
  2 = 96.0d                  for 0:10min
  3 =   0.9d/s to 50.0d
  4 = 50.0d                  for 0:05min
  5=    0.9d/s to 60.0d
  6 = 60.0d                 for 4:00min
  7 = Goto1 , 27 times
  8 =    0.9d/s to  4.0d
  9 =    4.0d                 for ever
10 = End

         d/s = degrees centigrade per second
         d    = degrees centigrade
xx:yymin = xx minutes plus yy seconds

The actually observed temperature ramp (MJ Research) is 0.53 till 0.84d/s ;
so it never exceeds the temperature ramp of the 9600 , 9700 or 2400 perkin
elmer machines (maximal 1.0d/s)
Enjoy your research
Gys de Jongh http://dermatology.azn.nl/lab/

Sequencing wrote in message <7h2fbn$mlc at net.bio.net>...
>
>I am inquiring as to whether anyone is successfully using a MJ Research
>PTC100 for cycle sequencing.  We are performing 10ul and 20ul reactions
with
>BIG DYE chemistry and are obtaining very low yield for our extension
>products.
>
>MJ Research has explained their cycler is very different from the 9600 - it
>begins timing after the block itself has reached temperature and does not
>take into account the time required for samples to reach temperature.  I
>have tried adjusting the times and still have not been successful.
>
>Has anyone has experienced similar problems with non-PE cyclers?  I would
>appreciate any advise.
>
>Thanks very much,
>
>Monette Aujay
>Rigel, Inc.
>






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