In vitro transcription reaction

1 ml linearized DNA template pBS-SK- ERD (X-E) (0.1-0.5 mg)

7.8 ml DDW

5 ml 5X transcription buffer ( stratagene )

1 ml 750 mM DTT

1 ml Rnasin (RNase inhibitor; 15U/ml)

1 ml 10 mM ATP (pH 7)

1 ml 10 mM CTP (pH 7)

1 ml 10 mM GTP (pH 7)

1 ml 1 mM UTP (pH 7)

5 ml (a-32P )-UTP (400-800 Ci/mmol; 10 mCi/ml) {final conc. 4-8 mM}

0.2 ml T7 or T3 or SP6 RNA polymerase (50 U/ml, Stratagen)

25 ml, mix, spin incubate at 37 oC for 30 min. Start: End:

Add

1ml DNase 1(free from Rnase -10 U/ml; BMB)

26 mL ,, mix, spin incubate at 37 oC for 15 min. Start: End:

End the reaction by adding:

7 ml 10X STE (0.2 M Tris-HCl, pH 7.5; 1 M NaCl; 0.1 M EDTA)

38 ml DDW

Mix by pipeting --Spot 1 ml on paper disk inside a scintillation bottle.

--Purify the rest probe on gel filtration PUSH COLUMN (Stratagene) .

Purification of unincorporated radiolabelled nucleotides from RNA probe using push gel filtration column

1- Wet Stratagene push column (#400701, 400700) with 80 ml of STE buffer (20 mM Tris-HCl, pH 7.5; 0.1 M NaCl; 10 mM EDTA).

2- Load transcription reaction on the column. Place screw cap tube to collect the radioactive probe. Pipette on top of the column 70 ml more STE and collect into the same tube. Mix and measure volume (~110-130 ml)

Calculate radioactive incorporation

Spot 1 ml of the eluted probe on paper.

Add to paper disk 4 ml scintillation cocktail ; read in scintillation counter.

(B)= CPM/1 X 70 ml = B- CPM

(A)= CPM /1 X elution Vol. (ml)= A- CPM

A-CPM/B-CPM X100 =% incorporation ### Usually 40-50% incorporation

specific activity - usually 1-5 X10 9cpm/mg RNA

3- The probe is ready for hybridization.

Pre-hybridization/ Hybridization conditions for RNA as a probe

Note- start labeling RNA probe after 1 h pre-hybridization .

A- Set up the temperature in hybridization incubator. 62 oC

B- Place the dry membrane (up to 4) in a hybridization screw cap tube.

Pre-hybrid. Stock solution ml [final concentration]

DECP DDW 0.8

Deionized formamide 5 50 %

20X SSPE 2.5 5X

50X Denhardts 2 10 X

10 mg/ml (3 min fresh boiled )salmon sperm DNA 0.1 100 mg/ml

20% SDS 0.1 0.2%

Final Volume 10

1- Pre-heat buffer to 62 oC add salmon sperm. Pre hybridize at 62 oC for 4 h.

2- Add RNA probe to final conc. of 5X10 6cpm/ml to the pre-hybridization sol.

Hybridize for 12 to 18 h, at 62 oC .

3- After hybridization Pour the hybridization solution to a radioactive waste .

Remove excess radioactivity by washing membranes twice with 25 ml of 2XSSC;0.5% SDS at room temperature.

Wash membranes at high stringency: 3X 15 min in 50 ml of 0.2X SSC, 0.5 % SDS at 62oC.

4- Place membrane on Whatman paper. Spot radioactive or fluorescent marker on the membrane. Cover with Saran wrap. Place intensifying screen, expose to Kodak XAR film at -70 oC for 2-3- days.