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 ^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 waterSuperScript 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, pH5.2Absolute ethanol80% 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 buffer8ul 5mM methyl dNTPs4ul 100mM DTT2ul oligo dT primer/adaptor (4ug)1ul [a-32P] dATP (~400Ci/mmol)1ul RNasin5.5ul H₂O10ul mRNA0.5ul 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 Buffer15ul 100mM DTT12ul 5mM dNTPs293ul sterile H₂O1ul [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.