Download Datasheet - Innosieve

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Pipetting the PCR mastermix and DNA samples
Table 2: Demonstration of the composition (per PCR reaction)
Components
RT-PCR premaster (white)
RNase free water (blue)
Volume / reaction
12,5 µl
11,0 µl
End concentration
-
UNG (yellow)
Probe (green)
Mix volume
DNA
Total volume
0,2 µl
1,0 µl
24,7 µl
1,0 µl
25,7 µl
0,2 Unit
variable
-
1. Depending on your total PCR amount pipette the adequate volumes of
premaster, water, Uracil-Glycosylase (UNG) and probe in a sterile
PCR tube (e.g.: 1,5-ml reaction cup).
2. Mix the mastermix on a shaker, to disperse components homogeneously.
3. Put the PCR components back into the freezer (at - 20 °C).
4. Aliquot 24 µl of your mastermix into the labeled PCR tubes (0,2 ml tubes, 8module-strips, or 96/384-PCR-plate). Don’t forget controls!
5. Pipette 1 µl of your sample DNA per cavity into the mastermix. This avoids
crosscontaminations!
6. Don’t forget to pipette 1 µl of the negative control (blue cup) and 1 µl of the
positive control (red cup) into separate PCR mastermixes. Include a positive
control reaction in your DNA preparation to exclude effects of inhibition by the
matrix analysed.
7. Close the tubes or plates well and put them into the thermocycler.
Thermocycling
Start the specific PCR program (see table 1, page 6).
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