Hat Conversion RACE Experiment
Hat Conversion RACE Experiment
The method presented here uses a biotinylated primer to help purify the first strand product. These purification techniques are interchangeable. Similarly, using the oligonucleotide-(dT)-based primers presented here, the gene-specific primers used at the start of reverse transcription (as described above) are also interchangeable. This experiment was derived from PCR Laboratory Guide (Second Edition) by Seed Kang and Qu Lijia.
Operation method
Hat Conversion RACE Experiment
Materials and Instruments
Oligonucleotide Primers cDNA Library HerculesHot-Start Polymerase HerculesHot-Start Polymerase Buffer dNTP Solution TE Reverse Transcription Buffer RNasin Superscript II Reverse Transcriptase Biotin-labeled Primers Ptotal Hat Discovery Junction Primer poly(A)+RNA Move reagents For more product details, please visit Aladdin Scientific website.
Thermocycler Streptomyces anti-biotin protein magnetic beads Magnetic separator Water bath or heater
The success of 5' RACE depends on the cap finding the splice sequence bound at the start of cDNA synthesis. This step relies on attaching an additional oligonucleotide (dC) to the end of the first strand cDNA. It has been shown that the efficiency of this step is greatly improved by the presence of Mn2+ in the reverse transcription buffer (SchmidtandMueller1999).
Phase 1: Reverse transcription to generate the cDNA template
I. Materials
1. buffers, solutions and reagents
dNTP solution (containing 4 dNTP, each 10mmol/L)
TE (10 mmol/L Tris-HCl, pH 7.5, 1 mmol/LTA, pH 8.0), preheat to 50°C
2. Enzyme and enzyme buffer
Reverse transcription buffer, 5X (250 mmol/LTris-HCl, pH 8.3 at 45°C, 30 mmol/LMgCl2, 10 mmol/LMnCl2, 50 mmol/LDTT, 1 mg/ml BSA)
RNasin
Superscript II reverse transcriptase (Invitrogen)
3. Nucleic acids and oligonucleotides
Biotin-labeled primers Ptotal (biotin-labeled primers can be ordered from Invitrogen.) The sequence of the Ptotal primers is shown in Figure 25-6.
Hat Discovery Junction Primer (1 mmol/L). The sequence of this primer is shown in Figure 25-6.
poly(A )+RNA
4. special equipment
Streptavidin anti-biotin protein magnetic beads (e.g. Promega's Streptavidin MagneSphere particles)
Magnetic separators (e.g., Promega's MagneSphere Magnetic Separation Stand)
Water bath or heater preset to 42°C, 45°C, 50°C, 70°C, 80°C 
Methods
1. In a sterile centrifuge tube, mix the following transcription components on an ice bath.
Reverse transcription buffer, 5X 4ul
dNTP solution (10 mmol/L) 0.4ul
Hat discovery splice primer (1 mml/L) 1ul
RNasin (40U/ul) 0.25ul
2. In another tube, add lug of poly(A)+RNA and 10pmol of Ptotal primer in 12ul of water, incubate at 80°C for 3 min, cool quickly on ice, centrifuge for 5s in a centrifuge, and add the reverse transcription components from the first step.
3. Add 1ul (200U) of Superscript II reverse transcriptase, incubate at room temperature for 5 min, 42°C for 30 min, 45°C for 30 min, and then 50°C for 10 min.
4. Incubate at 70°C for 15 min to inactivate the reverse transcriptase and isolate the first strand of cDNA using Streptavidin Magnetic Beads according to the manufacturer's instructions, using 5 times the amount of Streptavidin Magnetic Beads than the amount of biotinylated primers used. Wash the beads 3 times with TE at 50°C to remove excess capsid discovery junctions (according to the manufacturer's instructions).
5. Dilute the reaction mixture to 0.5 ml with TE and store at 4°C (this is the cDNA library).
Phase 2: Amplification of cDNA
I. Materials
1. buffers, solutions and reagents
dNTP solution (containing 4 dNTP, 10 mmol/L each)
2. enzymes and enzyme buffers
Hercules Hot-Start polymerase (Stratagene)
HerculesHot-Start Polymerase Buffer (10X)
Other hot-start PCR systems can also be used, such as Roche's Expand High Fidelity System.
3. Nucleic acids and oligonucleotides
Biotinylated cDNA library (from phase 1)
Oligonucleotide primers GSP1 and Po (for 3'RACE), or RGSP1 and Uo (for 5'RACE), sequences of Po and Uo are shown in Figure 25-6.
4. Specialized equipment
Programmable Thermal Cycler
5. Other
If a second round of amplification is required, the following reagents are needed in steps 5 and 6.
Primers GSP2 and Pi (for 3' RACE), or RGSP2 and Ui (for 5' RACE).The sequences of Pi and Ui are shown in Figure 25-6.
TE (10 mmol/LTris-HCl, pH 7.5, 1 mmol/LTris-HCl, pH 8.0)
II Methods
1. First round
(1) Mix the following components in a sterile 0.2 ml centrifuge tube.
Hercules Hot-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 cDNA library (resuspend magnetic beads well) and 25pmol of GSP1 with Po primer (for 3'RACE), or 25pmol of RGSP1 with Uo primer (for 5'RACE).
(3) Heat at 98°C for 5 min on a thermal cycler to denature the first strand product and streptavidin protein and activate the polymerase. Cool to the appropriate temperature and anneal for 2 min and extend for 40 min at 72°C. It is not necessary to keep the streptavidin magnetic beads in suspension during the incubation process because biotin cannot interact with denatured streptavidin. Styrene beads are reported to be smaller and can be kept in suspension without agitation and are therefore more advantageous (Schrammet al. 2000).
(4) Perform 30 amplification cycles according to the following procedure. 
The 72°C extension time must be adjusted according to the size of the product and the amplification rate of the polymerase used.
2. Second round (if required)
(5) Take a portion of the amplification product from the first round and dilute it with TE at 1:20.
(6) Using the procedure of the first round, but omitting the 2 min annealing and 40 mm extension step at 72°C, amplify 1ul of the above diluted material with either the GSP2 and P1 primers (for 3'RACE), or the RGSP2 he U1 primer (for 5'RACE).