In vitro transcription with yeast nuclear extract

Steve Hahn

Last Modified Fri,Apr 25,2003

Wear gloves throughout,use RNAse free solutions (either autoclaved or sterile filtered)and clean bench and pipetmen with 95% ethanol before use to eliminate any stray RNAses .

1.For a 25 microliter transcription reaction,add the following to a microfuge tube (when doing multiple reactions,it is easiest to make a larger mix and aliquot to individual reaction tubes):

5 microliters 5x transcription buffer

0.63 microliters 0.1 M DTT

3 microliters phospho creatine (64 mg/ml pH 7.5)

0.1 microliter creatine phospho kinase

1 microliter NTP mix (10 mM each ATP,CTP,GTP,UTP

150 ng plasmid DNA template

(0.125 microliters 10% NP40 can be added if desired but is not necessary)

10 units RNAse inhibitor (Amersham ~105 units/microliter)

24 ng Gal4-VP16 (0.2 microliters 0.12 mg/ml)or 30 ng Gal4-AH (0.3 microliters 0.1 mg/ml)

H20 to a final volume of 20 microliters (the final volume can be more or less than 20 microliters depending on the amount of protein to be added).

(add RNAse inhibitor,creatine phosphokinase,and activator protein to the mix last after all other components have been added and mixed well)

2.After addition of all components,mix well and aliquot to individual tubes at room temperature.Set up the reaction so that the Gal4-activator has at least 10 min to bind the template before the nuclear extract is added.

3.Depending on the volume of extract to be added to each reaction,add buffer HA + 0.1 M potassium acetate to each tube so that the final volume of the reaction will be 25 microliters (e.g.if 2.5 microliters of extract will be added,add 2.5 microliters HA + 0.1 M KOAc).Mix well and quick spin tubes.This addition of HA + 0.1M can be omitted if the same volume of extract will be added to all tubes.

4.Add yeast transcription extract directly to reaction and mix well by gently tapping the tube 5-6 times.Incubate 30-45 min at room temp.(typically add between 60 - 120 micrograms yeast nuclear extract per reaction.The optimum amount depends on the extract used and the response is not always linear with amount of extract added).

5.After the 30-45 min incubation,add 180 microliters stop mix.

6.Extract 1x with phenol/chloroform (2/1).Carefully transfer the aqueous layer to new tubes.

7.Ethanol precipitate by adding 1/10 volume 3 M sodium acetate and 3x vol ethanol.Wash pellets with 80% ethanol.Dry in speedvac.Assay RNA synthesis using primer extension.