Final concentrations of components in a 25 ul transcription reaction.

32.5 mM Hepes pH 7.6 (KOH adjusted)
50 mM KCl
6.25 mM MgCl
0.5 mM DTT
0.05 mM EDTA
5% Glycerol
5 ug/ul Drosophila embryo extract (extracts are typically 15 mg/ml)
10 ng/ul template DNA
0.5 mM each of 4 rNTPs

Notes: You can reduce the DNA concentration, as we do when we reconstitute pausing, but you then need to add carrier DNA to reduce nonspecific binding by proteins.  HaeIII-cut E.coli DNA is an effective carrier.  When working with dilute proteins, as might be the case with purified factors, addition of 2% polyvinyl alcohol can stimulate the reaction.

Assemble the components on ice.

In one tube, combine:

0.5 ul of 1 M Hepes pH 7.6.
2.5 ul of each 5 mM ribonucleotide triphosphate stock (adjust volume if stock concentration differs from 5 mM).
ddH₂O so final volume is 12.5 ul.

(Note that this can be assembled as a master mix if appropriate.)

In a second tube, prepare 12.5 ul of an appropriate ratio of extract and 0.1 M HEMG (0.1 M HEMG = 0.1 M KCl, 25 mM Hepes pH 7.6, 12.5 mM MgCl₂, 0.1 mM EDTA, 10 % glycerol).

Add the protein mixture to the first tube, mix thoroughly by triteration (not vortexing) and incubate at 25^oC for 30'.

Stop the reaction by adding 100 ul of transcription stop, mix thoroughly (Note that the subsequent manipulations are derived from the Kadonaga procedure.  I've selected this over the Biggin procedure because Kadonaga uses primer extension to detect the transcripts.)

Transcription stop (store at room temperature):

20 mM EDTA pH 8
0.2 M NaCl
1% SDS
0.25 ug/ul yeast RNA
(Do not use tRNA.  Based on information from the GeneX course and on of Kadonaga's papers, a good carrier RNA is prepared in the following way.  Dissolve Torula Yeast [type VI from Sigma] at 10 mg/ml in water.  Exhaustively extract the RNA: 3 times with phenol, 3 times with phenol/chloroform/isoamyl alcohol, and 2 times with chloroform.  Finally, ethanol precipitate the RNA to remove traces of chloroform.  Dissolve the RNA in TE.  Determine the concentration by measuring the absorbance.)

Add 1 ul of 10 mg/ml proteinase K and incubate at room temperature for 5 minutes.

Add 300 ul 0.3 M sodium acetate pH 6 and 400 ul phenol/chloroform/isoamyl alcohol.

Mix vigorously for 1 minute, microfuge 5', transfer the upper phase to a new tube.

Add 400 ul chloroform/isoamyl alcohol (24:1), mix vigorously, microfuge 5', transfer upper layer (400 ul) to a new tube.

At this point, the RNA can be stored at -70^oC, although there is no guarantee that it won't suffer some nucleolytic attack.

Add 0.03 pmoles of ³²P-labeled primer (prepared by kinasing and Nuc-trap purification) to the 400 ul of RNA and mix.  (Kadonaga's procedure indicates that about 1 ul of primer is added per sample.  This will vary depending on the primer preparation but be certain the primer has been diluted enough so that equal amounts of primer can be reproducibly added to each sample.  An alternative is to make a master mix with the 0.3 M sodium acetate used to dissolve the RNA described two steps ahead.)

Add 1 ml of Ethanol, cap tube and mix by inversion. Microfuge samples at room temperature for 15' and remove as much supernatant as possible.

Dissolve RNA pellet in 100 ul 0.3 M sodium acetate pH 6.  Add 250 ul Ethanol, mix by inversion and microfuge for 15' at room temperature.  Remove as much supernatant as possible.

Add 0.3 ml of 75% Ethanol, mix, spin 5' and remove supernatant.

Allow the pellet to dry.

Dissolve the pellet in 10 ul of 1 x annealing buffer.  This buffer consists of 2 mM Tris-Cl pH 7.8, 0.2 mM EDTA,  250 mM KCl (I filter sterilized my preparation.)

Place the samples in a 1 quart tub containing 2/3 quart of water heated to 75^oC.  Allow the water to cool to 37^oC before proceeding to the next step. (As might be expected, different primers have different optimal annealing conditions.  The GeneX manual suggests testing annealing temperatures of 37, 45, 60 and 68^oC rather than using the slow cool method described here.  The samples are incubated at the desired temperature for 90 minutes.)

Microfuge the samples briefly at 20' intervals to consolidate the liquid at the bottom of the tube.

At the end of the annealing, place the samples on ice.

Perform the primer extension reaction by adding 40 ul of extension mix and incubating the samples at 37^oC for 1 hour.

Extension mix per sample:
34.5 ul water
1.25 ul 1 M KCl
0.5 ul 1 M DTT
0.15 ul 1 M MgCl₂
2.5 ul 1 M Tris-Cl pH 8.3
0.25 ul 10 mM dGTP
0.25 ul 10 mM dCTP
0.25 ul 10 mM TTP
0.25 ul 10 mM dATP
15 Units of  M-MLV Reverse Transcriptase, RNase H minus (200 U/ul Fisher/Promega)

Note that the completeness of the extension reaction can be influenced by the sequence and secondary structure of the RNA.  Kadonaga uses a significantly different extension mix which might overcome extension problems - see me if you want details.  Different reverse transcriptases can work better than others (The GeneX manual recommends avoiding Boehringer Mannheim, M-MLV reverse transcriptase has lower RNase H activity than AMV-RT).  If you use a reverse transcriptase other than M-MLV, be certain the buffer conditions for the extension reaction are appropriate for the enzyme. Finally, actinomycin D is suppose to help overcome problems caused by RNA secondary structure - Kadonaga uses it at 125 ug/ml and I used to use it at 50 ug/ml.  However, earlier tests indicate that actinomycin D has no effect on the outcome with the hsp70 promoter.

Add 5 ul of 3 M sodium acetate pH 6 and 150 ul of Ethanol.  Mix and place on ice for 15 minutes.  Centrifuge for 15' and discard the supernatant.  Wash the pellet with 100 ul of 75% ethanol, spin 5' and remove as much supernatant as possible.

Thoroughly dry the samples and dissolve in sequencing loading buffer. (Note, Kadonaga's loading buffer consists of 3 ul of 0.1 M NaOH mixed with 6 ul of formamide loading buffer.  It occurred to me that boiling the sample in this buffer should lead to extensive RNA hydrolysis; hence, the NaOH might help eliminate spurious bands on the gel. Don't use the NaOH if you have used a different transcription protocol and your RNA contains the radioactivity)

Analyze on an 8% sequencing gel.