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PCR for PDH

Contributed by Dr. Alexei Gratchev

Amplification of 102 bp fragment from PDH cDNA and about 200 bp from genomic DNA (Acc.#: XM_113395).
Forward primer: 5'-GGT ATG GAT GAG GAG CTG GA, bases from 142 to 161.
Reverse primer: 5'-CTT CCA CAG CCC TCG ACT AA, bases from 243 to 224.
Reaction conditions
buffer 10x without Mg 2.5 µl
Mg (25mM) 1.5 µl
dNTP mix (2mM each) 2.5 µl
Primer forw (10mM) 1.0 µl
Primer rev (10mM) 1.0 µl
Taq polymerase (5U/µl) 0.2 µl
Template DNA (0.1-1µg/µl) 1.0 µl
H2O 15.5 µl

Cycling conditions for MJ Research DNA Engine
Initial denaturing 94° C 40 sec
30 cycles 94° C 40 sec
55° C 40 sec
72° C 1 min 30 sec
Final extension 72° C 5 min

This PCR is useful for analysis of DNase digestion as well as cDNA synthesis efficiency.

  1. Total RNA (contamination with genomic DNA detected)
  2. DNase treated total RNA
  3. cDNA
  4. Genomic DNA

Picture was kindly provided by Dr. J.-H. Martens, University of Heidelberg.
\METHODS\PCR\PCR for PDH
© Copyright 1999-2006 Alexei Gratchev. All rights reserved.