ISOLATION OF GENOMIC DNA FROM BUCCAL SWABS USING PHASE LOCK GEL

Day 1

1. Use a sterile cytology brush to scrape cells from the inside of each cheek (approximately 1 minute per cheek). Place the swab head in a 2.0mL tube containing 650µL of lysis buffer.

Note: Swabs can be kept in lysis buffer for an extended period of time before digestion and extraction are performed.

Day 2

4. Pour the sample into a 2ml phase lock "light" tube (Green) containing an equal (or excess) volume (650µL) of saturated, pH adjusted (pH 8.0) phenol. Mix by rocking gently.

Note: Keeping the swabs in the tube makes separation of aqueous phase slightly more difficult, but has been shown to increase yield.

5. Centrifuge 5 min at maximum speed to separate the phases.

6. Pour the aqueous phase into a 2 ml tube of phase lock "heavy"� gel (Yellow) containing an equal volume (650µL) of CHCl3:IAA (24:1)..

7. Centrifuge 5 min at maximum speed to separate the phases.

8. Pour the aqueous phase into a new tube.

9. Add 1/10 vol. of 3M NaOAc, and mix by rocking gently. Add 0.6 total volume of Isopropanol (cold if possible). Invert the tube several times to precipitate the DNA.

Note: It is also possible to precipitate using 1/2 volume of 7.5M ammonium acetate and 1 volume 100% ethanol. The yield will decrease with the age of the ammonium acetate.

10. Incubate at 0oC (on ice) overnight.

Note: This is an optional incubation that has been shown to increase yield

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Day 3

11. Pellet DNA by centrifugation at maximum speed 30 min.

12. Carefully decant the liquid and rinse the pellet with an excess (1000µl) of 70% EtOH (cold if possible). Mix by rocking gently.

13. Pellet the DNA by centrifugation at maximum speed for 5 min.

14. Carefully decant the EtOH and rinse the pellet a second time with an excess (1000µl) of 70% EtOH (cold if possible). Mix by rocking gently.

Note: This is an optional wash

15. Pellet the DNA by centrifugation at maximum speed for 5 min.

16. Carefully decant the EtOH and rinse the pellet with an excess (1000µl) of 95% EtOH (cold if possible). Mix by rocking gently.

17.  Pellet the DNA by centrifugation at maximum speed for 5 min. Carefully decant the EtOH, then dry the DNA in a vacuum chamber with little or no heat overnight.

18. Resuspend DNA in 50µl Low TE pH 8.0

REAGENTS

 
Lysis Buffer (with 1/2 x sucrose to reduce the density for better phase lock separation)

50 mM Tris pH 8.0

50 mM EDTA

25 mM Sucrose

100 mM NaCl

1 % SDS

10 mg/ml Proteinase K

Low TE pH 8.0

10 mM Tris pH 8.0
0.1 mM EDTA pH 8.0

95% EtOH

Phenol equilibrated with Tris pH 8.0

CHCl3:IAA (24:1)

Omni Swabs: Whatman Scientific Cat #WB10 0004

Eppendorf Phase Lock Gel 2ml Light Cat # 955154037

Eppendorf Phase Lock Gel 2ml Heavy Cat # 955154045

hogl organic extraction.pdf

Created by Matt Kaplan; 18 May 2004