Control Reactions with ProtoScript® M-MuLV Taq RT-PCR Kit

Protocol

1. 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

2. Mix well by pipetting and incubate at 42°C for one hour.
3. Inactivate the reverse transcriptase by heating at 80°C for 5 minutes.
4. 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

5. Analyze 5 μl of the reaction on a 1% agarose gel, stained with ethidium bromide.