This protocol is divided into three phases. Phase 1: reverse transcription to generate the cDNA template; phase 2: addition of poly(A) tail to the end of the first-strand cDNA product; and phase 3: amplification. This experiment is based on the PCR Laboratory Guide (2nd ed.) by Kang Seo and Lijia Qu.
Operation method
Experiment to amplify the 5' end of cDNA by classical RACE
Materials and Instruments
GSP-RT Primer poly(A)+RNA or total RNA Reverse Transcription Buffer RNAase H RNasin SuperScriptII Reverse Transcriptase dNTP Solution DTT TE CoCl2 dATP Solution Deoxyribonucleotide Terminal Transferase Tailoring Buffer HerculesHot-Start Polymerase HerculesHot- HerculesHot-Start Polymerase HerculesHot-Start Polymerase Buffer 5' end-spiked cDNA library Oligonucleotide primers Move Phase 1: Reverse transcription to generate cDNA template For more product details, please visit Aladdin Scientific website.
Thermal cyclers Water baths or heaters Microcon-100 filters
I. Materials
1. Buffers, solutions and reagents
dNTP solution (containing 4 kinds of dNTP, each 10mmol/L)
DTT (0.lmt)l/L)
TE (10 mmol/L Tris-HCl, pH 7.5, 1 mmol/LTTA, pH 8.0)
2. Enzyme and buffer
Reverse transcription buffer, 5X (supplied by manufacturer)
RNAase H
RNasin
SuperScriptII reverse transcriptase (Invitrogen)
3. Nucleic acids and oligonucleotides
GSP-RT primer (100ng/ul)
poly(A)+RNA or total RNA
4. Specialized equipment
Water bath or heater preset to 37°C, 42°C, 50°C, 70°C, 80°C
II. Methods
1. In a sterile centrifuge tube, mix the following transcription components on an ice bath.
Reverse transcription buffer, 5X 4ul
dNTP solution (contains 4 dNTP, 10 mmol/L each) 1ul
DTT (0.lmol/L) 2ul
RNasin(40U/ul) 0.25ul
2. In another tube, mix 0.5ul of GSP-RT primer (100ng/ul) and lug poly(A)+RNA or 5ug of total RNA, and add 13ul of H20. incubate at 80°C for 3 min, cool quickly on ice, and centrifuge for 5s.
3. add the RNA/primer mixture to the reverse transcription component, then add 1ul (200U) of SuperscriptII Reverse Transcriptase, mix gently, incubate at 42°C for 1h, then at 50°C for 10min.
4. incubate at 70°C for 15 min to inactivate reverse transcriptase, then centrifuge for 5s.
5. Add 0.75ul (1.5U) of RNAase H to the tube and incubate at 37°C for 20 min to disrupt the RNA template.
6. Dilute the reaction mixture to 40ul with TE and store at 4°C (this is the 5' end untailed cDNA library). 
Phase 2: Add poly(A) tail to the end of the first-strand cDNA product.
I. Materials
1. Buffers, solutions and reagents
CoCl2 (25 mmol/L)
dATP solution (1 mmol/L)
TE (10 mmolTris-HCl, pH 7.5, 1 mmol/LEDTA, pH 8.0)
2. Enzyme and enzyme buffer
Deoxynucleotide terminal transferase (Tdt)
Tailoring buffer, 5X (125 mmol/L Tris-HCl, pH 6.6, lmol/L potassium dimethylarsinate, 1250ug/ml BSA)
3. Special equipment
Microcon-100 filter (Millipore) or other products that serve the same purpose.
Water bath or heater preset for 37°C, 65°C
Methods
1. Remove the excess primer from the 5' end of the tailless cDNA library (obtained from Step 2 of Phase 1 above) using a Microcon-100 filter (Millipore) or other product that serves the same purpose, follow the instructions, and wash the library more than twice with TE, do not exceed a total volume of 15ul, and adjust the total volume to 15ul with water.
2. Add 4ul of 5X tailing buffer and 10U of Tdt.
3. Hold at 37°C for 5 min, then incubate at 65°C for 5 min.
4. Dilute volume to 500ul with TE and store at 4°C (this is the 5' tailing cDNA library). 
Phase 3: Amplification
I. Materials
1. Buffers, solutions and reagents
dNTP solution (containing 4 dNTP, 10 mmol/L each)
TE (10 mmol/L Tris-HCl, pH 7.5, 1 mmol/LTTA, pH 8.0)
2. Enzyme and enzyme buffer
Hercules Hot-Start polymerase (Stratagene)
HerculesHot-Start Polymerase Buffer (10X)
3. Nucleic acids and oligonucleotides
5' end-spiked cDNA library (obtained from phase 2 above)
Oligonucleotide primers Qo, QT, and GSPl (see Figure 25-2 for sequences of Q0 and QT)
4. Specialized equipment
Programmable Thermal Cycler
II. Methods
1. First round
(1) Mix the following components in a sterile 0.2 ml centrifuge tube.
HerculesHot-Start Polymerase Buffer (10X) 5ul
dNTP solution (10 mmol/L) 1.0ul
Hercules Hot-Start Polymerase 2.5U
H20 to 50ul
(2) Add 1ul of 5' end-spiked cDNA library (from Step 4 of Phase 2) and 25pmol each of GSP1,Q0 [see Figure 25-2(b)] and QT primers.
(3) Mix and heat at 98°C for 5 min on a DNA thermal cycler to denature the first strand product and activate the polymerase. Cool to 48°C and anneal for 2 min, then extend at 72°C for 40 min.
(4) Perform 30 amplification cycles according to the following program. 
2. Second round
(5) Remove a portion of the first strand amplification product and dilute with TE at 1:20.
(6) Amplify 1ul of the above diluted material with the Q1 primer and the GSP2 primer, using the above procedure but omitting the 2 min annealing and 40 mm extension at 72°C. 