Author: Unkonwn Date: Unknown

PCR Transgene Screening

Prepare Tail DNA

1. Prepare eppendorf tubes with 500 ul tail extraction solution each. Can be stored at -20.
tail extraction solution

20% SDS 5ml
1M Tris (pH 8.3) 5ml
0.5M EDTA 20ml
Proteinase K (20mg/ml) 5ml
dH20 65ml
100ml
2. Cut 1/2 cm of the tail off using a sharp razor blade. Place tip into tube with extraction solution.
3. Place in a 55oC water bath, overnight.
4. vortex, then do either A) or for "cleaner" DNA do B):

A)
1. microfuge the tubes at high speed for 10min.
2. transfer the sup to a clean tube, add 1ml 100%EtOH and vortex.

3. repeat spin at max speed for 10min.
4. carefully draw off sup, and allow pellet to dry.

5. Add 100 ul of ddH2O and resuspend

B)
1. microfuge the tubes at high speed for10 min.
2. transfer the supn. to a clean tube, add 500 ul of phenol (50mM Tris saturated phenol), vortex and spin for 1 min.
3. transfer the supn. to a clean tube, add 250ul of phenol and 250ul of chloroform, vortex, spin for 1 min.
4. transfer the supn. to a clean tube, add 500 ul chloroform, vortex, and spin 1 min.
5. transfer the supn. to a clean tube, add 50 ul of 3M Sodium acetate and 500 ul of isopropanol, vortex, spin for 10 min.

6. remove supn. add 250ul 70% EtOH to pellet, spin for 10 min.
7. Remove alcohol and dry pellet, resuspend the pellet in 100 ul TE and store at 4oC.