Laboratory: Unit 3: prepare genomic DNA (53-54)

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Laboratory Unit 3 prepare genomic DNA
(53-54) Lecture Genomic DNA purification In-Cla
ss Writing discuss JBC 266 3811-14 (1991)
(page 155) Hand In flow chart 3 lab report
2 Read AEM 63 2647-2653,
1997 (minus abstract) (see page
68) Due Next Class abstract for AEM 63 2647-53
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Use one isolate. Pick a dense culture.
Transfer 1 ml culture to sterile vial 0.2 ml
DMSO shake to mix. Label with your seat
number. Wrap tape around vial so ends overlap.
Place in freezer box. Place remaining culture
in 1.5-ml tube purify genomic DNA (section D).
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Purify Genomic DNA 1. Transfer 1.5 ml culture to
centrifuge tube. 2. Centrifuge maximum
speed 1 minute. Discard supernatant
(autoclave). 3. Suspend cells in 450 ml of
Tris EDTA disperse pellet
completely. EDTA chelates Mg ? inhibits
nucleases helps disrupt cells.
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4. Add 20 ml lysozyme, 20 min, 37oC. Lysozyme
cleaves peptidoglycan cell wall. 5. Add 10 ml
proteinase K, 20 min, 50oC. Proteinase K cleaves
cell wall proteins nucleases.
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6. Add 20 ml 25 SDS, 10 min, 68oC. SDS ionic
detergent disrupts membrane lipids denatures
proteins. 7. Add 57 ml 5 M NaCl vortex 1
minute. DNA RNA precipitate in high
salt/ethanol.
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8. Incubate 5 min, 68oC vortex 1 minute. Shears
DNA reduces viscosity. 9. Add 0.5 ml
phenolchloroformisoamyl alcohol (25241)
equilibrated with 1 M Tris. Mix well. Avoid
contact with organic solvents work in fume
hood. Lab coats, gloves, goggles, closed shoes
required. If phenol contacts skin, wash with
water seek medical attention. 10. Centrifuge
maximum speed, 5 minutes.
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11. Transfer aqueous (top) phase to 1.5-ml tube
with 0.5 ml chloroformisoamyl alcohol (241).
Withdraw top phase leave cell debris at
interphase. DNA attached to membrane will tug
at interphase. Discard organic solvents in
organic waste. 12. Mix well centrifuge maximum
speed, 2 min.
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13. Transfer aqueous (top) phase to 1.5-ml tube.
Leave cell debris at interphase. Discard
organic solvents in organic waste. 14. Add 1 ml
ethanol (ice cold), mix thoroughly hold 15 min,
0oC or 4oC. 15. Centrifuge maximum speed, 5
min orient tube in rotor so pellet forms at
known location. Discard supernatant solution.
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16. Wash DNA/RNA pellet with 0.5 ml 70 ethanol.
Let stand 1 minute do not disturb pellet.
17. Centrifuge maximum speed, 2 minutes.
Discard supernatant solution carefully pellet
will not adhere tightly to tube.
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18. Centrifuge 10 seconds. Remove ethanol with
pipet tip. Air dry pellet. 19. Dissolve
DNA/RNA pellet in 50 ml DNA buffer. Freeze at
-20oC.