Editing Phage DNA Isolation (section)

=== Extract DNA from Phage<br>  ===

<br> 

1. After precipitate phage using PEG/NaCl, resupend phage with 5 ml P1 with RNAse, add 5 ul benzonase, incubate at 37 <sup>o</sup>C for 1 hour.<br> 

2. Add 5 ml P2 (for plasmid maxiprep) , mix by votex, incubate for 10 min.<br> 

3. Add 5 ml of P3 (for plasmid maxiprep).<br> 

4. Incubate on ice for 2 min.<br> 

5. Spin at 13000 rpm for 2 X 2 min to pellet the white precipitate.<br> 

6. Collect supernatant to another tube. <br> 

7. Add equal volume (~15 ml) of isopropanol. Incubate on ice for 5 min.<br> 

8. Spin at 13000 rpm for 10 min to precipitate DNA.<br> 

9. Wash pellet with 70% ethanol, add 1 ml 70% ethanol, scrape DNA from centrifuge wall and transfer to a microcentrifuge tube, spin to pellet the DNA <br> 

10. Dissolve DNA in 500 ul TE.<br> 

11. Add 500 ul phenol-chloroform, mix by votex. 

12. Spin at 13000 rpm for 2 min. 

13. Collect supernatant to another tube. 

14. Add 500 ul isopropanol, incubate on ice for 5 min. 

15. Spin at 13000 rpm for 10 min. 

16. Decant liquid. Wash pellet with 100 ul 70% ethanol. Spin briefly, remove liquid. 

17. Add 100 ul TE to dissolve DNA. 

18. Check DNA quality with nanodrop, if you see a lot of RNA, run phage DNA on agarose gel and purify phage band using spin column. Elute DNA with 100 ul TE.<br> 

19. Digest phage DNA with HindIII and run digested and undigested DNA on gel.<br> 

2012/12/25: 

T4 Phage DNA after phenol-chl clean up. 

[[Image:T4 Phage DNA 2012 12 25.jpg]][[Image:T4 phage DNA.JPG]][[Image:T4 Phage DNA 2013 1 2.jpg]] 

[[Image:T4 phage DNA 2013 1 6.jpg]]<br>