Preparation of Phage DNA
Michael S. Parmacek
Reference: Maniatis plate lysate method 2.118
1. Inoculate 100 ul of phage stock into 300ul of Y1089 R- (if lgt11 phage) and incubate 20' at 37°C.
2. Add 7.5 ul of LB top agarose and plate on 150 mM LB Agarose plate.
3. Grow O/N at 37°C.
4. In the AM, plates should be completely blown away (individual plaques cannot be visualized).
5. Add 12 ml of SM (WITHOUT GELATIN) to each plate and gently shake at R.T for 90 minutes.
6. With a sterile pipette transfer supernatant to a 15 ml Sarstedt tube. Transfer 1 ml to another 15 ml Sarstedt tube and label as concentrated phage stock (add 1-2 drops of chloroform and store indefinately at 4°C).
7. Remove the bacterial debis by spinning 8500 RPM at 4°C for 10 minutes in a JA 17 rotor.
8. Pour off the supernatant into a second set of labelled Sarstedt tubes.
9. Add 10 ul of RNase A (1 mg/ml) and 10 ul of DNase I (1 mg/ml) to each tube and incubate 37°C for 30 minutes.
10. Add an equal volume (approximately 7-10 ml) of 20% PEG 8000, 2M NaCl in SM, invert tube several times and incubate 1 hr on ice (fill ice bucket with ice water slurry). Note: this will probably take two tubes.
11. Centrifuge 8500 RPM at 4°C for 20 minutes in a JA 13.1 rotor to recover the phage.
12. Pour off the supernatant, invert tube on a kimwipe and blot inside of the tube dry with a kimwipe. NOTE: THIS IS THE MOST IMPORTANT STEP YOU MUST GET RID OF ALL OF THE PEG OR THE DNA WILL NOT CUT WITH RESTRICTION ENZYMES.
13. Add 0.5 ml of SM to each tube and resuspend the phage by vortexing. Spin contents down (2000 RPM x 2 minutes) to get rid of debris and pool the tubes if necessary.
14. Transfer the resuspended phage to a microfuge tube and micrfuge at full speed for 2 minutes.
15. Transfer the supernantant to a second set of microfuge tubes and add 5 ul of 10% SDS and 5 ul of 500 mM EDTA. Inucube 68°C for 15 minutes to break open the phage.
16. Phenol/Chloroform x 2
Choroform/IAA x 1
17. Add 1/2 voume 7.5 M NH4Acetate and 2 volumes ETOH and precipiate O/N at -20°C.
18. Spin 5 minutes in a microfuge.
19. Wash with 1 ml of ice cole 70% ETOH. Spin 1 minute. Repeat wash. Speed vac to dry 2-3 minutes (do not over-dry).
20. Dissolve in 50 ul TE.
21 Set up restriction digests with approximately 3 ul of phage DNA (remember to add RNase to the digests).