1° cDNA Synthesis
N.B: During 1° cDNA synthesis, all steps should be carried while wearing gloves and all solutions should either have been DEPC-treated or purchased specifically for use with RNA.
You will need for 1° cDNA synthesis:
5 x SuperScript RTase buffer (Gibco-BRL)
5mM methyl dNTPs (Pharmacia, substitute 5mdCTP for dCTP)
100mM DTT (Gibco-BRL)
oligo dT primer/adaptor
[a-32P] dATP (~400Ci/mmol, Amersham or DuPont)
Ribonuclease inhibitor (RNasin, Pharmacia)
Sterile, autoclaved, DEPC-treated water
SuperScript RTase (or SuperScript II, Gibco-BRL)
You will need for 2° cDNA synthesis:
10 x DNA Polymerase Buffer (1M HEPES, pH 7.6, 40mM MgCl2, 2.5mM DTT, 675mM KCl)
100mM DTT (Gibco-BRL)
5mM dNTPs (Pharmacia)
Sterile, nano-pure H2O
[a-32P] dATP (~400Ci/mmol, Amersham or DuPont)
RNase H (Gibco-BRL)
DNA Polymerase I (Pharmacia)
3M sodium acetate, 100mM magnesium acetate, pH5.2
Absolute ethanol
80% ethanol
After isolation of mRNA, 1° cDNA synthesis is performed in the following way :-
1) Heat mRNA to 70°C for 10 minutes and snap chill on ice.
2) Add, in the following order, to a sterile, RNase-free, eppendorf tube:-
8ul 5 x SuperScript RTase buffer
8ul 5mM methyl dNTPs
4ul 100mM DTT
2ul oligo dT primer/adaptor (4ug)
1ul [a-32P] dATP (~400Ci/mmol)
1ul RNasin
5.5ul H2O
10ul mRNA
0.5ul SuperScript Reverse Transcriptase (~100 units).
Mix, incubate at 37°C for 1 hour and stop reaction by chilling on ice. Remove 4ul for analysis of the quantity and quality of the primary cDNA reaction products.
2° cDNA Synthesis
To the remaining 36ul of primary cDNA reaction products, ON ICE, add the following in the stated order:-
40ul 10 x DNA Polymerase Buffer
15ul 100mM DTT
12ul 5mM dNTPs
293ul sterile H2O
1ul [a-32P] dATP (~400Ci/mmol)
1ul RNase H (0.9 units)
2ul DNA Polymerase I (20 units)
Mix thoroughly and incubate for 1 hour at 14°C followed by 1 hour at room temperature (22°C). Place reaction on ice.
Extract once each with an equal volume of phenol/chloroform and chloroform. Precipitate cDNA products by the addition of 40ul 3M sodium acetate, 100mM magnesium acetate, pH 5.2 and 1ml absolute ethanol followed by incubation at -20°C overnight.
Pellet cDNA by centrifugation at 13,000 rpm at room temperature for 30 minutes. Wash pellet, VERY GENTLY, with 80% ethanol and vacuum dry. Re-dissolve pellet in 44.5ul sterile H2O and remove 4ul for analysis of cDNA quantity and quality.
The remaining 40.5ul is ready for blunt-ending.
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