Guidelines using ProtoScript First Strand cDNA Synthesis Kit
In order to avoid ribonuclease contamination use proper precautions, including the use of autoclaved tubes, baked glassware, ultra-pure solutions, sterile pipette tips and latex gloves during manipulations.
Thaw system components and put on ice. The 10X RT Buffer can be warmed briefly at 42°C and vortexed to dissolve any precipitate.
- In a sterile microfuge tube add:
Total RNA 1-10 μl (1 ng-2 μg)
Primer dT23VN 2 μl
dNTP Mix 4μl
Nuclease-free H2O to a total volume of
- Heat for 5 minutes at 70°C. Spin briefly and put promptly on ice.
- RNA/primer/dNTP mix from step 1 16 μl
10X RT Buffer 2 μl
RNase Inhibitor 1 μl
M-MuLV Reverse 1 μl
Final volume 20 μl
- Incubate at 42°C for one hour.
- Inactivate the enzyme at 95°C for 5 minutes.
If you wish to treat the cDNA products with RNase H (see FAQs), proceed to step 6, otherwise skip to step 7 or store at -20°C.
- Add 1 μl (2 units) of RNase H and inhibitor at 37°C for 20 minutes to degrade the RNA. Heat at 95°C for 5 minutes to inactivate the enzyme.
- Dilute reaction to 50 μl with water and use 2-5 μl for each PCR reaction.
A. Priming with Random Primers
If you are using random primers, use 2 μl of (dN)9 with total RNA amounts ranging from 10 ng to 1 μg. Outside this range the amount of (dN)9 needs to be increased or decreased respectively for optimal yield of cDNA.
B. Priming with a Specific Primer
If you wish to use a gene specific (anti-sense) primer, instead of one of the included primers, substitute DT23VN of (dT)9 with 15-20 pmol of your primer in step 1 of the first strand synthesis protocol.
C. Using poly(A)+ RNA
Use 1 to 100 ng of poly(A)+ RNA. The amount required depends on the abundance of transcripts in the sample.
D. Positive Control Reaction
Using the standard protocol, prepare cDNA from 1 μl (0.5 μg) total rat liver RNA. Use 5 μl out of 50 μl cDNA (step 7) for PCR. Follow the cycling protocol below using the control GAPDH primers included in the kit.
94°C 2 minutes
94°C 30 seconds
55°C 30 seconds } 25 cycles
72°C 30 seconds
Analyze the product (10-20% of the total PCR reaction) by agarose gel electrophoresis. A strong band of 350 bp should be visible by ethidium bromide staining (Figure 2). The included primers for GAPDH can be used for amplification of GAPDH from rat, mouse or human cDNA.