Worm Breeding for Super Geniuses: A Guide to Genetic Mapping in C. elegans
Synthesis of Double-Stranded cDNA from Purified Poly(A) mRNA 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 Procedure: 1. To 1.5mL RNase-free polypropylene tube, add: Volume final concentration 1. T7-(dT)24 primer 1ml 100pmol 2. mRNA 0.2 to 5 mg 3. DEPC-treated H2O enough to bring final volume (after step3) to 20ml 2.Add to tube: a. 5X first strand buffer 4ml 1X b. 0.1M DTT ...