cDNA AMPLIFICATION FROM LAMBDA-PHAGE LIBRARY
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1460
| PREPARE SOLUTIONS | |
| 1. SM buffer (1 L): |
Mix 5.8 g of NaCl, 2 g of MgSO4 -7H2 O, 50 mL of 1M Tris-HCl, pH 7.5, 0.5 mL of 2% gelatin, and dH2 O to 1 L (Autoclave)
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| 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 dH2 O
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| 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 4o C 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 | |
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4. Centrifuge for 5 minutes at 5,000 rpms. A gray pellet forms |
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| 5. Resuspend pellet in TENS buffer | |
| 6. Heat at 65o C for 10 mins | |
| 7. Extract with phenol followed by chloroform:isoamyl alcohol | |
| 8. Precipitate with isopropanol:sodium acetate | |
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9. Wash pellet with 70% ethanol |
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| 10. Resuspend pellet in T1 E0.1 , check DNA concentration and store aliquoted at -20o C |
