Reverse Transcription

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RT reaction for total RNA extracts

J.W. Jahng 2000

RNA (Trizol extraction)                       3ul (15ug)
oligodT-T7                                    1ul (100ng)
depc-dH2O                                   2.2ul 

heat up at 70 C for 10 minutes

chill on ice quickly

5 x first strand buffer                    5ul
100mM DTT                                2.5ul
10mM dATP                                  2ul
10mM dGTP                                  2ul
10mM dTTP                                  2ul
100uM dCTP                               1.2ul
32P-dCTP(40uCi/ul)                       2.5ul (100uCi)
RNasin                                   0.5ul

remove 0.5ul onto the filter paper “O”*

superscript                                 1ul

incubate the tube at 37 C for 2 hours.

remove 0.5ul onto the filter paper “F”*

Notes:

  1. when you prepare the filter papers for 10% TCA washing, cut them in the size of 7mm x 7mm each and write O(original) or F(after) on each filter with a lead pencil, and place the filters on a piece of parafilm, then leave them behind the epoxy shield.
  2. 10% TCA washing----use a top of the pipette tip boxes for P1000 filtered tips. Three washes for 5min, 5min, 30min each with gentle agitation by hands. When you change washes you can just pour out, the filters will stick on the plastic box. Place the filters in the scintillation vials and add the cocktail. TCA is dangerous, be careful not to burn your skin while you handle it!!!
  3. you can keep your RT tube in the refigerator if you’re gonna use it at the same day, otherwise keep it in the freezer.