ISOLATION OF SS DNA FROM BLUESCRIPT VECTORS USING HELPER PHAGE VCS-M13

ISOLATION OF SS DNA FROM BLUESCRIPT VECTORS USING HELPER PHAGE VCS-M13

Solutions:

• TES, pH 7.5; (20 mM Tris/20 mM NaCl/1 mM EDTA)
• 25% PEG-3M NaCl

1. From overnight plate or broth with antibiotics, inoculate 4 ml 2XYT or equivalent broth and grow to OD600 0.3
2. Add 40 ul VCS-M13 helper phage (titer ~1x10¹⁰ cfu/ml)
3. Grow with hard shaking 5-6 hr
4. Pellet in 1.5 ml microfuge tubes, 3 min
5. Remove 800 ul supernatant and add 175 ul PEG/NaCl; mix, and let stand 10 min at room temp (or ice overnight); centrifuge 5 min in microfuge
6. Pour off supernatant, dry inside of tube with Kimwipe
7. Add 100 ul TES, vortes 30 s
8. Add 100 ul phenol equilibrated with TE, vortex 10 s, spin 5 min
9. Repeat with chloroform extraction
10. Ethanol/sodium acetate precipitate; wash DNA pellet with 70% EtOH
11. Resuspend in 20 ul TE; 4 ul/sequencing reaction should be sufficient