( protocol given in the manual: Each 0.5...)Enhancing PCR* Efficiency with MasterTaq

protocol given in the manual: Each 0.5 l of a reverse transcription reaction was used as template in the PCR reaction containing 0.6 M 5' primer, 0.6 M 3' primer, 0.2 mM dNTPs, 1 unit Taq DNA Polymerase and 10 l 5x TaqMaster PCR enhancer in a total volume of 50 l 1 x PCR buffer including MgCl2. This reaction mixture was amplified using the following temperature program: 96C 2 min and 30 cycles of 96C 5 sec, 58C 5 sec, 72C 15 sec.

Results and discussion

Positive RT-PCRs for hsp27 and hsp60 could only be performed using the MasterTaq Kit. As shown in Fig. 1, hsp60 (312 bp) and hsp27 (291 bp) were amplified with MasterTaq (A). However, amplification was insufficient and non-reproducible with Taq from another supplier (B). After direct sequencing of the hsp60 and hsp27 amplicons, they were labeled with [....32P] dATP for use as probes in a Northern blot experiment (Fig. 2). Using this strategy, it was possible to detect hsp60 and hsp27 mRNA in human lung tumor cells.

Figure 1: RT-PCR of hsp60 (312 bp) and hsp27 (291 bp), 10 l each loaded onto a 1.5% agarose gel (stained with ethidium bromide).
Lane 1: 100 bp ladder (MBI Fermentas)
Lane 2: reverse transcriptase supplier 1/hsp60
Lane 3: reverse transcriptase supplier 2/hsp60
Lane 4: reverse transcriptase supplier 1/hsp27
Lane 5: reverse transcriptase supplier 2/hsp27

All reactions were of identical composition, except for enzyme and enhancer.

Series A: amplification using MasterTaq Kit (Eppendorf)
Series B: amplification using Taq Polymerase supplier M.
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