TITLE: Modified Phenol Chloroform Genomic DNA Extraction Protocol from the Christner Lab (University of Florida)
AUTHORS: Shelby  J Barnes, Noelle Bryan, Brent Christner, Cameron  Thrash

[DESCRIPTION]
Modified Phenol Chloroform genomic DNA Extraction Protocol from the Christner Lab (University of Florida) 

[STEPS]
SECTION: Lysis
1.  Start with filtered cells OR spin to get pellet from liquid culture.
SECTION: Lysis
1.1. Pipette off supernatant, if any.
SECTION: Lysis
2. Add 380 uL 1x TE Buffer + 20 uL Lysozyme to sample. 
SECTION: Lysis
3. Incubate for 1 h at 37˚C.
SECTION: Lysis
4. Add 60  uL SDS + 30 uL Proteinase K + 110 uL 1x TE  Buffer (total volume = 600 uL) to sample. 
SECTION: Lysis
5. Incubate for 2 h at 37˚C.
SECTION: Lysis
6. Vortex tube thoroughly. 
SECTION: Lysis
7. Incubate at 37˚C for 1 h , or until samples are clear. 
SECTION: Lysis
8. Incubate at 65˚C for 30 min to inactivate Proteinase K. 
SECTION: Lysis
9. Stop here and store at -20˚C  or move to extraction. 
SECTION: Phenol Chloroform Extraction
10. Add equal volume (600 uL) of Phenol Chloroform Isoamyl alcohol (P/C/I) to sample. 

SECTION: Phenol Chloroform Extraction
10.1. Invert samples thoroughly before centrifuge step. 
SECTION: Phenol Chloroform Extraction
11. Spin at 10,640 rcf for 10 min. 
SECTION: Phenol Chloroform Extraction
11.1. White interface should be visible. 
SECTION: Phenol Chloroform Extraction
12. Transfer aqueous phase (top layer) to new tube. 
SECTION: Phenol Chloroform Extraction
12.1. Transfer approx. 500 - 600 uL in 100 uL increments.

SECTION: Phenol Chloroform Extraction
12.2. Do not disturb interface. 
SECTION: Phenol Chloroform Extraction
13. Add equal volume of Chloroform Isoamyl alcohol (C/I) to sample. 
SECTION: Phenol Chloroform Extraction
13.1. Approx. 500 uL
SECTION: Phenol Chloroform Extraction
13.2. Invert sample thoroughly before centrifuge step. 
SECTION: Phenol Chloroform Extraction
14. Spin at 10,640 rcf for 10 min. 
SECTION: Phenol Chloroform Extraction
14.1. White interface should be visible. 
SECTION: Phenol Chloroform Extraction
15. Transfer aqueous phase (top layer) to new tube. 
SECTION: Phenol Chloroform Extraction
15.1. Do not disturb interface.
SECTION: Phenol Chloroform Extraction
15.2. Approx. 300 - 500 uL
SECTION: Phenol Chloroform Extraction
16. Store at -20˚C  or move to ethanol precipitation. 
SECTION: Ethanol Precipitation
17. Add 2 volumes of ice cold 100% Ethanol to sample.
SECTION: Ethanol Precipitation
17.1. E.g. Have 50 uL of DNA solution, add 100 uL Ethanol.
SECTION: Ethanol Precipitation
18. Add 0.1 volumes of 3M Sodium Acetate pH 5.2 to sample.
SECTION: Ethanol Precipitation
18.1. E.g. Have 50 uL DNA solution, add 5 uL Sodium Acetate.
SECTION: Ethanol Precipitation
19. Incubate at -20˚C  for at least 1 hr. 
SECTION: Ethanol Precipitation
19.1. Cold incubation for a longer period of time (e.g. over night) may lead to higher yield.
SECTION: Ethanol Precipitation
20. Spin at 13,000 rcf for 30 min at 4˚C . 
SECTION: Ethanol Precipitation
20.1. Should see small white pellet. 

If tube is placed hinge-side outward, the pellet should be found below the hinge and almost to the bottom. 
SECTION: Ethanol Precipitation
21. Pipette off supernatant. 
SECTION: Ethanol Precipitation
22. Gently add 150 uL of 70% ethanol to remove salts. 
SECTION: Ethanol Precipitation
22.1. Do not dislodge pellet. 

Keeping the samples on ice or in cold tube racks will help pellet stay in place. 
SECTION: Ethanol Precipitation
23. Spin at 13,000 rcf for 10 min at 4˚C . 
SECTION: Ethanol Precipitation
24. Pipette off supernatant. 
SECTION: Ethanol Precipitation
25. Spin at 13,000 rcf for 1 min to bring down any excess ethanol. 
SECTION: Ethanol Precipitation
26. Pipette off any remaining liquid. 
SECTION: Ethanol Precipitation
27. Dry pellet for 5 min MAX. 
SECTION: Ethanol Precipitation
27.1. Any longer makes resuspending difficult. 
SECTION: Ethanol Precipitation
28. Resuspend pellet in 50 uL Molecular Bio water. 
SECTION: Ethanol Precipitation
28.1. Use 1- 3 uL of DNA to quantify using Qubit HS dsDNA assay kit. 
SECTION: Ethanol Precipitation
29. Store at -20˚C .