Super ScriptII: 18064-014 (need own oligos, Rnase Inhibitor) 1/2002

Reverse Transcription Protocol FGB

Date:_______ Sample(s):___________________________ DNased________

Oligo(s) used: Oligo dt, Random Primers, or Gene Specific preimers:

DNase RNA if desired to remove any chromosomal DNA that may be there.

This is for a 1X (20ul) reaction. A 2X reaction often works best.

Run a minus RT control!

1. Add the following to a pcr tube for a 1X reaction:

per reaction: 4X reaction:

1-5 ug total RNA x ul

Oligo-dt primers 1 ul

(0.5 ug/ul)

or 1-5 ul (1-5 uM) of 3'oligo 1uM= 2 ul

or 1-5 ul random hexamers (50 ng/ul)*

10mM dNTP mix 1 ul ______

DEPC treated H2O

up to 12 ul y ul up to 48 ul

For all oligo’s do the following three steps:

2. Heat mixture to 65'C for 5 minutes to remove 2’ structure/DNase from above.

3. Chill on ice ASAP then spin down (if no prior DNase treatment since already done if DNased).

4. Add the following:

RT Reaction: per reaction: 4X reaction:

5X First Strand Buffer 4 ul _____

0.1M DTT 2 ul _____

1ul RNase out (40U/ul) 1 ul _____

(no MgCl2 as in 5X buffer)

4. Mix, and then spin down.

5. Incubate for 2 minutes at 42'C (for oligo dT and GSP)!

Or at 25'C for 2 minutes (for random primers only)!

Then add:

Super script (200U/ul) 1 ul _____

f.v.= 20 ul f.v.= 80ul

5. Incubate for 50 minutes at 42’C.

6. Inactivate the enzyme by incubating at 70’C for 15 minutes.

7. Treat the DNA with RNase;1 ul of E. coli RNase H, 2 units/ul at 37’C for 20 minutes.

8. Dilute cDNA as needed. Use 10 % of this reaction (100 ng) in each PCR reaction.