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Reverse Transcription

Reverse Transcription

#### Standard Operating Protocol (SOP) Written 20150702 by Sam White.

##### Reagents: - M-MLV Reverse Transcriptase (Promega: M1701) - Primers (oligo dT: Promega: C1101 OR random: Promega: C1181) - 10mM dNTPs (Promega: U1511)

##### Personal Protective Equipment (PPE): - Gloves

##### Equipment: - Pipettes (10 - 1000uL) - Filtered pipette tips - 0.5mL snap-cap microfuge tubes (Genesee: 22-281A) - Sterile 1.7mL snap-cap microfuge tubes (Genesee: 22-281S) - Thermal cycler, water bath, or heating block capable of 37C OR 42C. - vortexer - ice

#### Procedure ##### Total Time: ~ 1.5 - 2.0hrs ##### Cost/sample: ~ $1.50 IMPORTANT: A single reaction volume = 25uL. The volume of RNA, primer(s) and M-MLV RT used in this protocol are variable and will be specific to your current experiment. The directions below apply to a reaction using 1ug of total RNA. You may need to make changes to accommodate your own conditions.

  1. Read the manufacturer's protocol (PDF).
  2. Read this protocol.
  3. Verify sufficient quantities of reagents and samples before beginning.
  4. Wear clean gloves.
  5. Thaw all RNA and reagents on ice. Prepare all reactions on ice.
  6. Transfer 1ug of RNA to 0.5mL snap cap tubes or PCR plate. Adjust volumes of individual samples to 17.75uL with H2O.
  7. Add 0.25ug primer per 1ug of RNA in sample (= 0.5uL of Promega oligo dT Cat#C1101 in this example). Total volume (RNA + primers) should equal 18.25uL.
  8. Heat samples at 70C for 5 min in thermal cycler, heating block, or water bath.
  9. Place samples on ice IMMEDIATELY.

  10. Make Master Mix:

    Per Reaction

    • 5 uL 5x Buffer (M-MLV RT Buffer)

    • VORTEX THOROUGHLY TO DISSOLVE PRECIPTATE

    • 1.25 uL 10mM dNTPs

    • 0.5 uL M-MLV RT per ug of RNA

  11. Mix well by flicking; do not vortex.

  12. Add 6.75uL of master mix to each reaction.
  13. Mix by pipetting; do not vortex.
  14. Incubate @ 42C for 1hr for oligo dT primers OR @ 37C for random primers.
  15. Heat inactivate @ 95C for 3 min.
  16. Spot spin and store @-20C.