M13 Single-Stranded Template Preparation

1. Dilute an overnight culture of host cells (DH5a, JM101, NM522) 1:100 in 25 ml TB broth and infect with a single plaque of recombinant M13.
2. Shake vigorously for 6 hours at 37ºC.
3. Collect the supernatant by centrifugation at 12,000 x g for 15 minutes. Pour supernatant into a fresh tube and repeat.
4. Precipitate the phage by the addition of 0.25 volumes of phage precipitation solution and incubate on ice for 30 minutes, then centrifuge at 12,000 x g for 15 minutes. Thoroughly drain supernatant and wipe excess away with a Kimwipe.
5. Resuspend pellet in 400 ul TE.
6. Add 1 volume of 24:1 cholorform:isoamyl alcohol and vortex for 1 full minute. Spin at 12,000 x g for 5 minutes.
7. Transfer the aqueous phase to another tube; leave the interface behind. Add 1 volume of TE saturated phenol/chloroform, vortex for 1 minute, centrifuge at 12,000 x g for 5 minutes.
8. Repeat above step until there is no longer an interface present.
9. Transfer the aqueous phase to a fresh tube and add 0.5 volumes 7.5 M ammonium acetate, pH 7.5 and 2 volumes of 100% ethanol. Mix and incubate at -20 for 30-90 minutes.
10. Centrifuge at 12,000 x g at 4ºC for 15 minutes, rinse the pellet with 70% ethanol and dry at bench for 3-5 minutes.
11. Resuspend the pellet in 50 µl of deionized water.

Phage precipition solution:

• 3.75 M ammonium acetate
• 20% PEG-8000
• Add equal volumes of 40% PEG-8000 and 7% M NH4OAc, pH 7.5
• TE-saturated phenol/chloroform
• Mix equal parts of TE and phenol and allow the phases to separate. Then mix 1 part of the lower, phenol phase with 1 part chloroform:isoamyl alcohol (24:1).