cDNA AMPLIFICATION FROM LAMBDA-PHAGE LIBRARY |
|
Last Update: December 2006 |
|
|
|
| PREPARE SOLUTIONS | |
| 1. SM buffer (1 L): | Mix 5.8 g of NaCl, 2 g of MgSO4-7H2O, 50 mL of 1M Tris-HCl, pH 7.5, 0.5 mL of 2% gelatin, and dH2O to 1 L (Autoclave) |
| 2. TENS buffer (100 mL): | Mix 5 mL of 1M Tris-HCl, pH 8.0 (50mM), 20 mL of 0.5 mM EDTA (100mM), 2 mL of 5M NaCl (100mM), 3 mL 10% SDS (0.3%), and 71 mL of dH2O |
| PROCEDURE | |
| 1. Plate cells and phage as described (144. cDNA library screening) and let plaques develop | |
| 2. Pippet enough SM buffer on plates to barely cover the plate and rock at 4oC for >1 hour | |
| 3. Pippet solution containing phage into a tube and add 0.2 mL of 2M ZnCl2 per 10 mL of SM solution | |
4. Centrifuge for 5 minutes at 5,000 rpms. A gray pellet forms |
|
| 5. Resuspend pellet in TENS buffer | |
| 6. Heat at 65oC for 10 mins | |
| 7. Extract with phenol followed by chloroform:isoamyl alcohol | |
| 8. Precipitate with isopropanol:sodium acetate | |
9. Wash pellet with 70% ethanol |
|
| 10. Resuspend pellet in T1E0.1, check DNA concentration and store aliquoted at -20oC | |