Super Script II 9/2001

Reverse Transcription Protocol FGB

For SYBR green PCR

Using GibcoBRL’s: Random Primer or Gene specific primers

Cat No. 11904-018

Date:_______ Sample(s):___________________________ DNased________

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

This is for a 2X (f.v.= 40ul) reaction.

Use Oligo dT.

1. Add the following For a 2X reaction:

2X reaction: Master Mix:

1 ug total RNA (1ug/ul) 1 ul ______ add 1 ul per tube.

Oligo-dT primers 2 ul

(0.5 ug/ul)

10mM dNTP mix 2 ul ______

DEPC treated H2O 15 ul (up to 20 ul)

(For a 2X reaction) add 19 ul per tube.

Then,

2. Heat mixture to 65'C for 5 minutes.

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

4. Add the following:

RT Reaction: per reaction: Master Mix:

10X 1^st Strand 4 ul _____

React Buffer

25mM MgCl2 8 ul _____

0.1M DTT 4 ul _____

1ul RNase out 2 ul _____

18 ul total add 18 ul per tube.

Mix well, then incubate for 2 minutes at 42'C for oligo dT primers.

Then add:

Super script II (200U/ul) 2 ul _____ add 2 ul per tube.

f.v.= 20 ul

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

8. Go to SYBR PCR reaction. Use 10% of this reaction (100 ng) in each PCR reaction. Do so by diluting 2-3X with dH2O.