3' Rapid Amplification of cDNA Ends RACE Using PCR Protocol
Date Added: April 11, 2008 10:26:01 PM
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First Strand cDNA Synthesis Protocol
1. Dissolve 1 to 5 μg of poly (A)+ RNA or 10 to 20 μg of total RNA in 10 μl of DEPC-treated water (see Tip #2).
2. Incubate the RNA at 65°C for 5 min.
3. Chill the RNA on ice.
4. Add the following to the RNA:
2 μl of 10X PCR Buffer
2 μl of 10 mM dNTPs
2 μl of 10 mM DTT
0.5 μl of RNasin
3 μl of 100 ng/ml (dT)17-Adaptor
1 μl of AMV Reverse Transcriptase (see Tip #3)
5. Incubate the reaction at 42°C for 1 to 2 hr.
6. Incubate the reaction at 95°C for 5 min to stop the reaction.
7. Dilute the reaction mixture with 180 μl of TE
Rapid Amplification (RACE) of Target cDNA Protocol
1. Use 1 to 10 μl aliquots from the above cDNA pool and add the following:
3. Combine 10 μl aliquots of the PCR products with 5 μl aliquots of Sucrose Loading Dye.
4. Load the samples on an Agarose Gel for analysis of the reaction products (see Tip #6)
RACE Buffers
DEPC-ddH2O
DEPC-ddH2O
DEPC-ddH2O
Shake or stir the solution
Autoclave for 15 min to inactivate the DEPC (CAUTION! See Tip 1)
Also see Tip 2
Allow solution to sit for at least 12 hr
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.
2. To minimize degradation of RNA by RNases, wear gloves when handling samples and reagents, and change gloves regularly while working. Treat water and solutions with DEPC (diethyl pyrocarbonate) to inactivate RNases, and use solutions prepared with RNase-free water and equipment.
3. AMV Reverse Transcriptase is used because it is more stable at higher temperatures than other reverse transcriptases. This allows for the reaction to proceed at 55°C, if so desired, to eliminate potentially inhibitory RNA secondary structures.
4. There is no need to purify the cDNA because the RNase H activity of the AMV Reverse Transcriptase degrades the RNA template.
5. Cycle conditions will depend on your primer, the size of the PCR product, etc. Optimize the PCR for your particular application.
6. Southern Blot analysis can also be performed using an internal gene-specific probe.
