Control Reactions with ProtoScript® M-MuLV Taq RT-PCR Kit
- The following control reactions can be used to examine the quality of kit components and RT-PCR products produced 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 a PCR product is detected in the -RT control reaction, it is due to either the contamination of genomic DNA or a carry-over PCR product.
Positive Control -RT control 10X RT Buffer 2 μl 2 μl Murine RNase Inhibitor 0.5 μl 0.5 μl Rat Liver Total RNA (500 ng/μl) 1 μl 1 μl dT23 VN (50 μM) 2 μl 2 μl dNTP mix (2.5 mM) 4 μl 4 μl M-MuLV Reverse Transcriptase 1 μl - Nuclease-free H20 9.5 μl 10.5 μl Final volume 20 μl 20 μl
- Mix well by pipetting and incubate at 42°C for one hour.
- Inactivate the reverse transcriptase by heating at 80°C for 5 minutes.
- Dilute the cDNA by adding 30 μl H2O, 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 30 Cycles 94°C 30 seconds 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.