Section on Neural Gene Expression

Tail DNA Analysis

1. Place numbered Eppendorf tubes in dry ice.
   
2. Cut 0.5cm from end of tails and place in tubes. Store at -80°C until analyzed.
   
3. Add 0.3ml of STE/PK solution to the Eppendorf tubes.
   
4. Incubate at 55°C overnight with rocking or for several hours with occasional mild vortexing every 15min.
   
5. Add 2µl 10mg/ml RNase A solution.
   
6. Mix by mild vortexing and incubate 37°C for 30min.
   
7. Cool to room temperature. Vortex 20s and spin for 5min.
   
8. Save supernatant (if pellet not visible, repeat previous step) and add 0.3ml isopropanol (2-propanol). Mix by inverting tube 20 times.
   
9. Centrifuge for 5min. Discard supernatant. Rinse pellet with 0.3ml 70% EtOH. Spin briefly and pipette off remainder of EtOH.
   
10. Let air-dry for 30min, and add 50 µl TE. Leave at room temperature overnight or heat for 1hour at 65°C to dissolve.
    
11. Optional: measure OD₂₆₀ and use about 8µg DNA per lane if performing Southern analysis.
12. For PCR analysis, run thermocycler at (95°Cx1min, 63°Cx1min, 72°Cx1min)x30; 72°Cx5min; 4°C end for, say, 19-22-mer primer pairs that are less than 600 bases apart or (95°Cx1min, 72°Cx3-4min)x40; 72°Cx10min; 4°C end for, say, 48-mer primer pairs that are greater than 1500 bases apart. For the latter conditions, if DNA polymerase is added with a hot start, replenish the enzyme after about 1 hr.

SETPK ingredients:

Enter number of tail samples:
(and then click elsewhere)

4M NaCl: µl 1M Tris-HCl, pH7.5: µl 250mM EDTA, pH8: µl 10% SDS: µl 10mg/ml proteinase K: µl water: µl

The Section on Neural Gene Expression is in the Laboratory of Cellular and Molecular Regulation, Division of Intramural Research Programs is within the National Institute of Mental Health (NIMH) is a part the National Institutes of Health (NIH), and is a component of the U.S. Department of Health and Human Services.

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