synthesis of RNA in situ probes

Set up in vitro transcription reaction as follows:

Reagent	Volume
5x transcription buffer (ProMega):	4ul
10x hapten (Dig. Bio DNP) U-NTP mix	2ul
100mM DTT	1ul
RNase inhibitor (RNasin) (50 μ/μl)	2ul
linearized DNA (~250 ng if 0.2μg/μl)	1ul (vary according to conc.)
RNA Polymerase (T3, T7, or Sp6)	2ul
H2O	to 20ul

1. Incubate at 37°C for 3hrs
2. Test by running 1 uL on a gel (materials should be NaOH treated)
3. Add 30ul H2O
4. Add 50ul2x carbonate buffer
5. Mix and incubate at 65 C for 5’
6. Add 100 ul Stop solution
7. Check sample fractionation on a gel
8. Add 8 ul 4M LiCl
9. Add 600 ul EtOH
10. Mix and freeze at –20°C for > 30 min (Overnight is good)
11. Spin at 4°C and 14k 20 min
12. Wash pellet in 70% EtOH
13. Air dry pellet and resuspend in 200 ul hybridization mix
14. (or less according to transcription efficiency as determined by gel)
15. Store at –20°C, use at ~2.5:100 (e.g..: ~5ul in a 200ul over night hybridization)

Reagents:

DNP mix

• 250 nmol of DNP-11-UTP (Perkin Elmer)
• 100 mM ATP, GTP and CTP deoxyribonucleic acids
• 7 uL GTP + 7uL CTP + 7 uL ATP + 4.5 uL UTP + 250 nmol DNP-11-UTP,
• fill to 70 uL with ddH2O

2x carbonate buffer

• 120 mM Na[2]CO[3]
• 80mM NaHCO[3]
• pH to 10.2, aliquot, store at -20C

STOP soln

• 0.2M NaOAc
• pH to 6.0 with acetic acid
• aliquot, store at -20C