||This kit contains reagents for five double-stranded (ds) cDNA synthesis reactions and control primers for 5' and 3' RACE PCR. A thermostable DNA polymerase for PCR is not included in the kit. Preparation of poly(A)+ RNA is required*
A. First-Strand cDNA Synthesis
1. 100 ul 5X RTase (Reverse Transcriptase) Buffer
250 mM Tris-HCl, pH 8.3
375 mM KCl
15 mM MgCl2
2. 10 ul Oligo(dT)20 Primer (10 uM)
3. 5.5 ul M-MLV (Moloney Murine Leukemia Virus)
Reverse Transcriptase (Advanced Type) (50 U/ul)
Source: An E. coli strain carrying a recombinant plasmid
B. Second-Strand cDNA Synthesis
4. 200 ul 5X Second-Strand Synthesis Buffer
100 mM Tris-HCl, pH 7.5
500 mM KCl
25 mM MgCl2
50 mM (NH4)2SO4 (Ammonium Sulfate)
5 mM Dithiothreitol (DTT)
0.25 mg/ml Bovine Serum Albumin (BSA)
0.75 mM B-Nicotinamide Adenine Dinucleotide (B-NAD)
5. 5.5 ul E. coli RNase H (1 U/ul) Source: An E. coli strain carrying a recombinant plasmid
6. 5.5 ul E. coli DNA ligase (5 U/ ul) Source: An E. coli strain carrying a recombinant plasmid
7. 11 ul E. coli DNA polymerase I (12 U/ ul) Source: An E. coli strain carrying a recombinant plasmid
8. 300 ul EDTA (0.5 M EDTA, pH 8.0)
C. Other Reagents
9. 40 ul 10 mM dNTP Mix (10 mM each of dATP, dCTP, dGTP, dTTP)
10. 500 ul 7.5 M Ammonium Acetate
11. 1.7 ml Distilled Water, Deionized, Sterile
12. 1.6 ml TE, pH 8.0
10 mM Tris-HCl (pH 8.0)
1 mM EDTA (pH 8.0)
*BioMagR mRNA Purification Kit (Polysciences), NucleoTrapR mRNA (MACHEREY-NAGEL), FastTrackR 2.0 mRNA Isolation Kit
(Life Technologies), Absolutely mRNA Purification Kit (Agilent Technologies), etc. can be used for preparation of poly(A)+ RNA.
D. RACE PCR
Control 5* and 3* RACE Primers
13. 200 ul Mouse Transferrin Receptor (Tfrc) 5* RACE Primer (10 pmol/ul):
5*-TTCTCAGGTGGCAGCTTTGAACT-3* (Tm 62.58C)
14. 200 ul Mouse Transferrin Receptor (Tfrc) 3* RACE Primer (10 pmol/ul):
5*-CGTGGAGACTACTTCCGTGCTAC-3* (Tm 62.55C)
(Full-length cDNA ~4.9 kb)
Note: Kit components are arranged in a row from left to right according to the approximate order of use in an experiment.
In RACE (Rapid Amplification of cDNA Ends) by the single-primer method* of this kit, the targeted cDNA is amplified by PCR with only a gene-specific primer using ds cDNA as a template.
The mechanism is based on that the terminal region of the ds cDNA is partially denatured at 68 C for the extension reaction and that the linear DNA molecule tends to circularize.
Upon reaching the 5' end of the template DNA, thermostable DNA polymerase switches templates to the 5' terminal region of the newly synthesized daughter strand at a certain probability and synthesizes DNA sequences complementary to the gene-specific primer.
Using this daughter strand as a template, the targeted cDNA is amplified with only a gene- specific primer.
Feature and advantages
By using the accura-expRACE KIT, both 5' and 3' RACE can easily and efficiently be performed under simple conditions, such as RT-PCR.
Long cDNA and rare cDNA unidentified previously can be isolated.
The synthesized ds cDNA can be used as a cDNA library. You can perform ~400 screenings of a cDNA library by RACE.
The cDNA synthesized by this kit contains a high proportion of full-length cDNA because an "advanced type M-MLV Reverse Transcriptase" is used.
• Okayama, H. et al., ,Mol. Cell. Biol. 2, 161-170 (1982).
• Gubler, U. et al.,Gene 25, 263-269(1983).
• Frohman, M.A., et al., Proc. Natl. Acad. Sci. USA 85, 8998-9002 (1988)
• Chenchik, A. et al BioTechniques 21, 526-534. (1996)