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3&#39;-RACE PCR

互联网2013-11-13

1926

实验试剂

Sterile water (high purity, RNase-free)

5 x SuperScript RTase buffer (Life Technologies)

RNase inhibitor (Pharmacia)

SuperScript RTase (Life Technologies)

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

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.

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.

实验步骤

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

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°undefined 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.

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

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相关问答

问

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1 回答 1418 围观

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