3RACEPCR

實驗概要

        This technique is used to obtain the 3'end of a cDNA, it requires some sequence information internal to the mRNA under study. The sequence information obtained from this technique can be utilised to obtain full length cDNA clones using the 5'RACE technique.
        The method I've used is loosely based on that given in 'PCR protocols: A guide to methods and applications', Academic Press Inc.
        The protocol given uses SuperScript II reverse transcriptase (Life Technologies) and Taq polymerase from Boehringer Mannheim. You will need to adjust the reagents according to the enzymes you use.

主要試劑

    Sterile water (high purity, RNase-free) 

    5 x SuperScript RTase buffer (Life Technologies) 

    100mM DTT 

    2.5mM dNTPs 

    RNase inhibitor (Pharmacia) 

    SuperScript RTase (Life Technologies) 

    10 x PCR buffer (Boehringer Mannheim, contains 15mM MgCl2) 

    500mM dNTPs 

    Mineral oil 

    RNA : Both total and polyA RNA are suitable for this technique. I recently compared the two and found the polyA RNAreaction to have the edge, but  
                only just. I do recommend using polyA RNA for 5'RACE though. 

    Primers： 

                T17 Adapter primer (T17AP): GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT 

                Adapter primer (AP): GACTCGAGTCGACATCG 

                Gene specific primer: Designed from known sequence, ideally it should an 18-22-mer with an annealing temperature of around 56°C, where
                 a G/C = 4°C and an A/T = 2°C.

實驗步驟

1. Place approx. 1ug of total RNA (~10ng polyA RNA) in an RNase-free eppendorf, make up to 10ul with pure water. Add 1ul of T17AP (0.5ug/ul).

2. Heat this to 70°C for 10 minutes, then place on ice for 5 minutes.

3. Spin pulse the tube and then add the following reagents:

    4ul 5X SuperScript RTase buffer 

    2ul 100mM DTT 

    1ul 2.5mM dNTPs 

    1ul RNase inhibitor 

    1ul SuperScript RTase.

4. Mix and incubate at 37°C for 2 hours.

5. Use a serial dilution of the reverse transcription reaction: 2ul of undiluted cDNA, 2ul of 10 x diluted cDNA and 2ul of 100 x diluted cDNA in the PCR reaction. Set up the PCR as follows:

    35.5ul pure water 

    5ul 10 x PCR buffer (Boehringer Mannheim) 

    5ul 500uM dNTPs (some enzymes require a higher concentration of dNTPs (2mM), for example Pfu polymerase (though I'd recommend against the  
       use of this enzyme) 

    1ul Adapter primer (25uM) 

    1ul Gene specific primer (25uM) 

    2ul cDNA template (various dilutions)

6. Mix and overlay with mineral oil.

7. Perform the following PCR cycles:

    a. 94°C 3 minutes 

    b. 72°C 3 minutes (at this point add 0.5ul of Taq polymerase) 

    c. 94°C 30 seconds 

    d. 56°C* 1 minute 

    e. 72°C 2 minutes (longer if expected fragment size is greater than 2kb) 

    f. Cycle steps c. - e. 35 times 

    g. 72°C 10 minutes.

* Varies according to the melting temperature of your gene specific primer. The annealing temperature in the PCR may require some adjustment for optimal yield of PCR product.

8. Load all or part of the reaction on an agarose gel. Don't be too surprised if you get a faint background smear as well as your prominent band