Guidelines for PCR Optimization Using ProtoScript II RT-PCR Kit
We recommend 2-5 μl of the diluted cDNA product per 50 μl PCR reaction.
- Mix the following in a PCR tube on ice:
Taq 2X Master Mix 25 μl (mix well by inverting before use)
Forward Primer (10 μm) 1 μl (final concentration 200 nm)
Reverse Primer (10 μm) 1 μl (final concentration 200 nm)
Diluted cDNA 2-5 μl
Total volume 50 μl
- Mix gently. Overlay with mineral oil if the thermal cycler lacks a heated lid.
- The following PCR cycling conditions are recommended for 0.2 ml thin-wall PCR tubes on BIO-Rad icycler or similar thermocyclers. For other PCR tubes or cyclers, it may be necessary to modify the cycle times:
Initial Denaturation 95°C 1-2 minutes
25-35 cycles 94°C 30 seconds
45-68°C 10-30 seconds
68°C 1 minute per kb
Final Extension 68°C 5-10 minutes
- Analyze 5 μl of the PCR product by agarose gel electrophoresis.
- The following control reactions can be used to examine the quality of kit components and RT-PCR products by the kits. The positive control reaction should give a 327 bp fragment, and no product is detectable in the –RT Reaction (figure 3). If the PCR product is deteced in the –RT control reaction, it is due to either the contamination of genomic DNA or a carry-over PCR product (see troubleshooting guide).
Positive Control -RT Control
10X RT Buffer 2 μl 2 μl
RNase Inhibitor 1 μl 1 μl
Rat Liver Total RNA 1 μl 1 μl
dT23VN (500 μm) 2 μl 2 μl
dNTP mix (2.5 mM) 4 μl 4 μl
M-MuLV Reverse 1 μl —
Nuclease-free H2O 9 μl 10 μl
Total volume 20 μl 20 μl
- Mix well by pipetting and incubate at 42°C for one hour.
- Inactivate the reverse transcriptase by heating at 90°C for 5 minutes.
- Dilute the cDNA by adding 30 μl H20 and add the diluted DNA to the following PCR reaction:
Taq 2X Master Mix 25 μl
GAPDH Primer Set (10 μm) 1 μl
Diluted cDNA 2 μl
H2O 22 μl
Total volume 50 μl
The following PCR cycling conditions are recommended:
Initial Denaturation 94°C 2 minutes
94°C 30 seconds
30 Cycles 55°C 15 seconds
68°C 30 seconds
Final Extension 68°C 5 minutes
- Analyze 5 μl of the reaction on a 1% agarose gel, stained with ethidium bromide.