7 In Situ Hybridization for RNA: Radioactive DNA Probe
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*TNEB
- 20 mM Tris-HCl, pH 7.0 ( 0.2M pH 7.0, 5ml )
- 0.3 M NaCI (5M 3ml)
- 2 mM EDTA, pH 7.0 (0.2M Na2EDTA. pH 7.0, 0.5 ml)
- 2x Denhardt (SOx Denhardt 2ml, H20 8 ml)
20% dextran sulfate
- mix 2.5g dextran sulfate with approximately 3 ml of H20 in a
15 ml Corning centrifuge tube
- dilute to 5ml with H20
- boil for 10 mins.
- store at -20°C until use
TNEB-DS
- TNEB, 600 III
- 50% dextran sulfate, 400 III
1. Mix TNEB-DS and formamide (1 : 1) thoroughly using Vortex
2. Heat at 80 a C for 3 mins and chill in ice water
3. Spin for 1 sec and keep in ice water until use
1. Apply 20 III of the mixture on a pretreated slide
2. Cover the sample with a sterile 22 x 22mm cover glass carefully
so as not to make bubbles, and seal its edges with a 1:1 mixture
of rubber cement and petroleum ether. Dry the cement.
3. Incubate slides by placing them on an aluminum cake pan
floating on a water bath and cover with Styrofoam plate at
45°C for 18 to 20 hrs so as not to drop water on the slides.
1. Peel off rubber cement with fine forceps
2. 2x SSC: 1 hr at room temp. Remove cover glass
Note: Do not remove forcefully, the cover glass comes off in solution spontaneously
3. 2x SSC: 10 mins x3 at room temp.
4. 0.5x SSC*: 10 mins at 45°C *2 x SSC: 20 ml; H20: 60 ml
5. O.lx SSC*: 10 mins x3 at 45°C *2 x SSC: 12.5 ml; H20 up to
250 ml
Hybridization
Washing