| A Problem of TAIL PCR | | 点击： 作者：51protocol收集 来源： 时间： 2007-03-29 本站论坛 |
|  | 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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