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cDNA synthesis and labelling for yeast

• cDNA synthesis and labelling
• cDNA Purification

cDNA synthesis and labelling:

For the RT reaction, we use a PCR machine (mastercyclerEppendorf) for all the incubation steps.

For each condition (Cy3 and Cy5):

1. Primary mix :.
   In a 0.2ml PCR tube put :
• 10µg of your purified total RNA
• 5µg of random hexamers (Roche, ref :1034731)
• 2µg of oligo dT (Invitrogen, ref : yo1212)
• add bidistilled autoclaved water to a final volume of 23µl
2. Incubate 10min at 70°C.
3. Put immediately on ice.
   Add 1µl of Cy3 OR Cy5 (1mM) from Amersham-Pharmacia.
   Add 16µl of the following mix to each tube: for one double-labelling reaction (Cy3 tube and Cy5 tube),
   16µl of 5X Superscript II buffer (Gibco BRL, ref : 18064-014), 8µl of DTT (0.1M) (supplied with the Gibco BRL Superscript II kit), 4µl of dNTP mix (do it yourself : 2.5mM dATP, dCTP, dGTP, 1.25mM dTTP), 4µl of Superscript II from Gibco BRL (200U/µl).
4. Incubate 10min at 23°C
5. Incubate 2 hours at 42°C
6. RNA Hydrolysis: add 15ul of 0.1M NaOH (made just before use).
7. Incubate 10min at 70°C.
8. pH neutralisation: add 15ul of 0.1M HCl

cDNA Purification:

1. Pool the two reactions (Cy3 and Cy5).
2. Add 1/10th volume of Sodium Acetate 3M pH 5.2 and 2.5 volumes of 100% Ethanol.
3. Precipitate at –80°C for 30 minutes.
4. Centrifuge 30 minutes at 13500 rcf at 4°C
5. Pour off all ethanol carefully. You do not need to dry the tube.
   Proceed to the following extra-purification step using a Quiaquick column (Quiagen, ref : 28106) to reduce the background. This step is essential to avoid high background on your slide :
6. Resuspend pellet in 40µl of water, add 4µl of Sodium Acetate and 200µl of Quiagen buffer PB.
7. Apply the mixture to a Quiaquick column and centrifuge at 13,500 rcf for 1 minute.
8. Discard flow-through, add 600µl of PE buffer. Spin at 13,500 rcf for 1 minute.
9. Discard flow-through and spin for 2 more minutes at 13,500 rcf to dry filter.
10. Put the column in a new, clean, 1,5ml centrifuge tube.
11. Elute by adding 30µl of water preheated to 37°C, let stand 1 minute, spin for 1 minute at 13500 rcf. You can repeat this elution step with the same 30µl to ensure better yield of elution.