Mixtures

1. Ethanol series: 70%, 90%, 99% (v/v). Can be stored at room temperature, but sucks water from the air (changes concentration).

2. 0.1% (w/v) Pepsin in 0.1 MHCl: freshly prepared and preheated to 37°C (it may take several minutes to dissolve pepsin even at 37°C); optional.

3. RNase A mixture (optional): for 100 pL, mix 80 pL of H2O, 10 pL of 1 pg/pL RNase A, and 10 pL of PBS. Prepare fresh.

4. Restriction enzyme mixture (Roche): for 100 pL, mix 78 pL of H2O, 10 pL of 1 pg/pL BSA, 10 pL of 10X restriction buffer, and 2 pL of restriction enzyme (2 U/100 pL have proven sufficient for nick translation in situ (8), but we have often used more here—as much as 50U/100 pL mix). Prepare fresh.

5. ^-Exonuclease mixture (New England Biolabs): for 100 pL, mix 68 pL of H2O, 10 pL of 10X incubation buffer, 10 pL of 1 pg/pL BSA, 10 pL of concentrated glycerol, and 2 pL (10-20 U) ^-exonuclease. Prepare fresh.

6. Denhardt's solution (4X): 400 mg of Ficoll 400, 400 mg of polyvinylpyrrolidone (PVP), 400 mg of BSA (fraction V), and H2O to 500 mL. Filter sterilize and store at -20°C in 25-mL aliquots.

7. Hybridization mixture: for 100 pL, mix 12 pL of H2O, 25 pL of 4X SSC, 25 pL of 40% (v/v) formamide, 25 pL of 4X Denhardt's solution, 12 pL of 2 pg/pL salmon sperm DNA, and 1 pL of each relevant DNA probe (from 1 pM stock solutions). Prepare fresh.

8. Padlock removal solution: 2X SSC and 30% (v/v) deionized formamide.

9. T4 DNA ligase mixture, for 100 pL, mix 78 pL of H2O, 10pL of 1pg/pL BSA, 10 pL of 10X ligase buffer, and 2 pL of (10 U) T4-DNA ligase. Prepare fresh.

10. Reaction mixture for rolling-circle PRINS: for 100 pL, mix 56 pL of H2O, 10 pL of 10X polymerase buffer, 10 pL of 1 pg/pL BSA, 2 pL of 50mMDTT, 10 pL of concentrated glycerol, 10 pL of 10X dNTP (1X dNTP: 250 nM of each), 2 pL of 9-29 DNA polymerase. Prepare fresh.

11. Wash buffer: 4X SSC, pH 7.0, 0.05% (v/v) Tween-20. Store up to 1 yr at room temperature.

12. Blocking solution (optional): Dissolve 5% (w/v) nonfat dry milk in wash buffer. Centrifuge for 2 min in a microcentrifuge at maximum speed and use supernatant. This solution can be stored at -20°C for years. When in use, it is preferable to store the blocking solution at 4°C and not use it for more than 1 wk (the milk turns sour with time, and the same may happen to the milk powder if it sucks water from the air).

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