TAIL PCR

[Bosco, Simona]

Dear fellow Arabidopsis researchers

         I am going to use TAIL PCR for amplification of genomic sequences
flanking the T-DNA insertion. I would like to use the methode described in
the following source:

Liu et al., Plant Journal 8: 457-463 (1995)


In this paper the the program for the primary PCR is:

  File no.1   Cycle no.1
                  93 C, 30 sec; 62 C, 1 min; 72 C, 2.5 min

  File no.2       Cycle no.5
                  94 C, 30 sec; 62 C, 1 min; 72 C, 2.5 min

  File no.3       Cycle no.1
                  94 C, 30 sec; 25 C, 3 min, ramping to 72 C, over 3 min;

                  72 C, 2.5 min

  File no.4       Cycle no.15
                  94 C, 10 sec; 68 C, 1 min; 72 C, 2.5 min
                  94 C, 10 sec; 68 C, 1 min; 72 C, 2.5 min
                  94 C, 10 sec; 44 C, 1 min; 72 C, 2.5 min

  File no.5       Cycle no.1
                  72 C, 5 min

I have a problem with the PCR machine. In the Liu`s paper they use the
Perkin-Elmer GeneAmp PCR System 9600 model.
  I am trying to set the same PCR program on the Perkin-Elmer GeneAmp PCR
System 9700 model but the PCR machine does not accept 1 cycle only for the
file no.3. The minimun number of cycles the machine accepts at this step is
2.

  Do you know anybody had used the original protocol setting 2 cicles instead
of 1 at this step?

  Thanks for your help
  Simona Bosco
  Dept. of Biological Sciences,
  University of Essex


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