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Making an in situ probe

1.  Linearize Qiagen-purified plasmid with appropriate blunt or 5’ overhang enzyme.

2.  Add 2 µl of Proteinase K (10mg/ml), 37^°C 1 hr

3.  Phenol/Chloroform extract digest and precipitate with 100% EtOH

4.  Resuspend pellet in DEPC-treated or RNase-free H₂0 (to a concentration of 1-2 µg/µl)

DNA	5 µl
RNase-free H20	8 µl
10X buffer	2 µl
Dig NTP mix (Roche kit)	2 µl
T7, T3, or SP6 (Roche)	1.5 µl
RNase Inhibitor (Roche)	0.5 µl
	20 µl

5.  Synthesis of probe:

6.  Run above 20 µl reaction 3 hrs at 37^°C

7.  DNase treatment:  Add 2 µl to reaction, return to 37^°C for 20 min

8.  Stop reaction:  Add 2 µl to reaction

9.  Precipitation of probe:

To 24 µl reaction, add: -20°C cold 4M LiCl                 2.5 µl -20°C cold 100% EtOH           75 µl Place reaction in –80°C 40 min Spin max speed, 4°C                15 min Pour off supernatant Add –20°C cold 70% EtOH     300 µl Spin max speed, 4°C                15 min Pour off supernatant

10.  Remove excess EtOH with pipette (do not vacuum dry) resuspend RNA in 20 µl DEPC-treated or RNase-free H₂0; gently mix

11.  Add 0.5 µl of RNase Inhibitor to RNA.  Proceed to run gel and spot test probe. 

12.  Store at –20^°C

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