The phage lysate from the plate contains bacterial DNA and RNA, as well as phage DNA encased in the phage coat. The following procedure, digests bacterial RNA and DNA, precipitates the phage particles, then removes the phage coat with organic extraction. Finally, the DNA is purified by precipitation with alcohol and visualized by gel electrophoresis.
Protocol
1. Add RNase A and DNase I, each to a final concentration of 1mg/mL, into 10 mL of plate lysate of phage in a 30 ml Corex tube
2. Incubate the lysate with nuclease at 37oC for 30 minutes.
3. Add 4 ml of Precipitant Solution: 33% PEG (polyethylene glycol), 3.3 M NaCl and mix them well.
4. Incubate the mixture on ice for 1 hr and then spin at 8,500 rpm for 10 minutes at 4oC.
5. Pour off the supernatant and dissolve the pellet in 0.6 ml of CSM and transfer it to a new 1.5 ml tube.
6. Add equal volume of the organic solution: phenol:chloroform:isoamyl alcohol 3:1:0.05. Mix them well by vortexing.
7. Spin the mixture at 14,000 rpm for 10 minutes at room temperature.
8. Transfer the upper aqueous layer to a new 1.5 ml tube and repeat steps 6 and 7 three times.
9. Transfer the upper aqueous layer to a new 1.5 mL tube. Add equal volume of chloroform and mix well.
10. Spin the mixture at 14,000 rpm for 10 minutes at room temperature.
11. Transfer the upper aqueous layer to a new 1.5 mL tube and add equal volume of isopropyl alcohol.
12. Mix well and incubate at -80oC for 12 minutes and spin at 14,000 rpm for 20 minutes at room temperature.
13. Dispose of the supernatant and add 0.7 mL of 70% ethyl alcohol.
14. Spin the tube at 14,000 rpm for 10 minutes.
15. Discard the supernatant and dry the pellet in the vacuum for 10 minutes.
16. Dissolve the pellet in 50 mL of TE buffer.
17. Load 10 mL of your sample on a 0.7% agarose gel and electrophorese it.
18. Take a picture of the gel.
19. Analyse the gel and present tables of the results and the photograph.
