àUse T7-(dT)24 oligomer for priming first-strand cDNA synthesis:
5’-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)24-3’
High-quality HPLC-purifed T7-(dT)24 primer should be used (NOT PAGE-purified primer). This is available from GENSET.
1st strand synthesis
starting material: 0.2-5mg poly (A) mRNA
àdetermine correct volumes of H2O and reverse transcriptase to be used: for every mg of RNA, need 1ml of SSIIRT (200U/ml). For mRNA quantity < or = 1mg, use 1ml of SSII RT.
Procedure:
2.Add to tube:
a. 5X first strand buffer 4ml 1X
b. 0.1M DTT 2ml 10mM
c.10mM dATP, dCTP,
dGTP, dTTP 1ml 500mM each
transcriptase 1ml per mg mRNA 200U to1000U
FINAL VOLUME FOR REACTION: 20ml
DEPC-treated water 91ml
5X second strand reaction buffer 30ml 1x
10mM dATP, dCTP, dGTP, dTTP 3ml 200mM each
10U/ml DNA ligase 1ml 10U
10U/ml DNA polymeraseI 4ml 40U
2U/ml RNase H 1ml 2U