3' Rapid Amplification of cDNA Ends RACE Using PCR Protocol

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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

DEPC-ddH₂O		0.1% (v/v) Diethyl Pyrocarbonate (DEPC) in ddH₂O

Adaptor		50 ng/μl Adaptor 5'-GAC TCG AGT CGA CAT CG-3'

10 mM dNTPs		10 mM dATP 10 mM dTTP 10 mM dGTP 10 mM dCTP

Gene-specific Primer		50 ng/μl anti-sense primer to gene of interest

10X PCR Buffer		15 mM MgCl₂ 500 mM KCl 100 mM Tris-Cl, pH 8.3

DEPC-ddH₂O

(dT)17-Adaptor		5'-GAC TCG AGT CGA CAT CGA TTT TTT TTT TTT TTT TT-3' 100 ng/μl (dT)17-Adaptor

Sucrose Loading Dye (6X)		0.25% (w/v) Xylene Cyanol 0.25% (w/v) Bromophenol Blue 40% (w/v) Sucrose

2 mM dNTPs		2 mM dGTP 2 mM dCTP 2 mM dATP 2 mM dTTP

10 mM DTT

Materials for RACE

AMV Reverse Transcriptase
dCTP
RNasin
dTTP
dGTP
Sucrose
dATP
Magnesium Chloride
Isoamyl Alcohol
Chloroform
Phenol
Potassium Chloride
Tris
Potassium Cacodylate
Diethyl Pyrocarbonate (DEPC)
DTT
Cobalt Chloride
(dT)17-Adaptor
DNA Polymerase, Taq
Bromophenol Blue
Oligonucleotide
Xylene Cyanol

Protocol Tips

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.

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