Library cDNA Synthesis

 

Library cDNA Synthesis

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 ^5m dCTP 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 H₂ O
[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 H₂ O
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 H₂ O
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 H₂ O and remove 4ul for analysis of cDNA quantity and quality.

The remaining 40.5ul is ready for blunt-ending.