Tag Archives: FISH441

Reverse Transcription – FISH441 RNA

Reverse transcribed the class FISH441 RNA to cDNA. Followed protocol provided on the FISH441 Wiki page, NOT the usual Roberts Lab protocol, with some modifications.

Changed total reaction volume to 50uL to accommodate volume of RNA, as well as low concentration of dNTPs that were to be used.

RNA – 17.75uL

Oligo dT – 1.0uL

Samples were heated at 70C for 5mins and placed immediately on ice.

Reaction buffer master mix calcs:

5x MMLV Buffer – 10uL/rxn X 88 = 880uL

2.5mM dNTPs – 10uL/rxn X 88 = 880uL

M-MLV – 1uL/rxn X 88 = 88uL

H2O – 9.25uL x 88 = 902uL

Added 31.25uL of the reaction buffer master mix to each well of RNA/Oligo dT. Reaction was run at 42C for 1hr, followed by 70C for 3mins. Samples were stored @ -20C in Rm. 209.