Peqlab peqSTAR series Kurzanleitung Seite 8

Short Manual peqSTAR thermal cyclers
05
Temp
06
Temp
07
Alternative programming of steps 03 – 07:
03
Start Cycle
04
Temp
05
Temp
06
Temp
07
Close Cycle
08
Temp
09
Store
PEQLAB_v1212E
Annealing at 65°C in the 1
'Touchdown'-PCR via Temperature-'Decrement'. The annealing temperature of 65 °C in the
st
1 cycle is reduced by 0.4 °C each cycle. This leads to high specifity in the first cycles and
high amplification efficiency in the later cycles.
The reduction of the cooling rate at this step of the program is not necessary, but should
only be an example. It might make sense e.g. for quantitative annealing of complementary
oligo nucleotide or at annealing steps in the first cycles, when primer annealing should be
very efficient because of low template amount (e.g. after cDNA synthesis)
Elongation at 72 °C for 1 minute in the 1
Via Time -'Increment' the elongation time is prolonged by 5 s each cycle. The incremental
prolongation of the elongation time is useful with high numbers of cycles (>30), when the
Polymerase activity declines.
Terminate 'Cycle' function with 'Close Cycle'.
Open program cycle – 30 cycles.
Denaturation at 95 °C for 30 seconds
Annealing at 65°C in the 1
'Touchdown'-PCR via Temperature-'Decrement'. The annealing temperature of 65 °C in the
1 st cycle is reduced by 0.4 °C each cycle. This leads to high specifity in the first cycles and
high amplification efficiency in the later cycles.
The reduction of the cooling rate at this step of the program is not necessary, but should
only be an example. It might make sense e.g. for quantitative annealing of complementary
oligo nucleotide or at annealing steps in the first cycles, when primer annealing should be
very efficient because of low template amount (e.g. after cDNA synthesis)
Elongation at 72 °C for 1 minute in the 1
Via Time -'Increment' the elongation time is prolonged by 5 s each cycle. The incremental
prolongation of the elongation time is useful with high numbers of cycles (>30), when the
Polymerase activity declines.
Terminate program loop with 'Close Cycle.
Final elongation at 72 °C for 5 minutes
The final elongation assures the complete synthesis of all amplificats.
8 °C forever, lid heat is turned off
By the 'Store' step the lid heat is turned off automatically. This avoids that lid heat and
Peltier elements of the block work against each other. Especially at longer incubation times
at low temperature this increases the life time of the Peltier elements and reduces the power
consumption.
7
st
cycle for 30 seconds
st
cycle
st
cycle for 30 seconds
st
cycle