IGEM:PennState/2007/Dnaprep

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Current revision (14:17, 22 June 2007) (view source)
 
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Miniprep of Bacterial Genomic DNA
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<h2>Telt Method -DNA Plasmid Preperation</h2>
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note: This method is "not very good" in that it does not seperate plasmid DNA from chromosomal DNA.  The advantage of this is that if there is no plasmid, you will get the chromosomal DNA.  Use this prep to seperate the chromosomal DNA when there is no plasmid in the cell.
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#Grow bacterial strain to saturation
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*Use sterile techniques throughout
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#Spin 1.5 mL for 2 min in microcentrifuge
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*all centrifuge steps are at room temperature
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#Resuspend in 567 ul TE buffere, 30ul of 20 mg/ml proteinase K. Mix and incubate 1 hr at 37C
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*put a tube of 100% ethanol on ice at start of procedure
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#*http://www.neb.com/nebecomm/products/productP8102.asp
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#Add 100ul of 5M NaCl. Mix thoroughly
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#Grow cells overnight in 3mL LB (with appropriate antibiotics and sugars) in a 13 x 100mm tube at 37C (or other appropriate temperature) in a rotator.
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#Add 80 ul of CTAB/NaCl solution. Mix. Incubate 10 min at 65C.
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#Pour 1.5 mL into sterile microcentrifuge tube. Centrifuge cells for 30 sec. Remove supernatent by aspiration
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#*CTAB/NaCl solution (0.7M NaCl, 10% CTAB): Dissolve 4.1 g NaCl in 80 ml water. Slowly add 10 g CTAB. Stir with heat to dissolve. Bring volume to 100 ml.
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#Resuspend cells in 100uL TELT buffer. Vortex to resuspend pellet.
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#Extract with an equal volume of chloroform/isoamyl alcohol. Spin 5 min in microcentrifuge.
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#Add 100uL 1:1 Phenol/Chloroform.
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#Transfer aqueous phase to a fresh tube. Precipitate DNA with .6 vol isopropanol. Wash precipitate with 70% ethanol. Remove supernatant and briefly dry pellet in lyophilizer.
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#Vortex for 15 sec. (Can leave cells for 5 minutes on th ebench).
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#Resuspend pellet in 100ul TE buffer.
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#Centfigue for 1min. Transfer top layer to clean tube.
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#Add 100uL chloroform/isoamyl (24:1)
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#Vortex for 15 sec. (Can leave cells for 5 minutes on th ebench.)
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#Centrifuge for 1 min.
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#Transfer 75uL of the supernatent to a new microcenttrifuge tube.
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#Add 200uL of 100% EtOH (cold). Mix well by inversion. Incubate at room temperature 2-10 minutes.
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# Centifuge for 10 minutes.
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#Pour off supernatant.  Add 1mL 70% EtOH. Invert several times. Pour off liquid. Spin briefly to bring liquid to bottom of tube. Remove rest of liquid by aspiration, being careful not to disturb pellet.  
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#Air dry pellet (about 1/2 hour). or vacuum dry 5 minutes.
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#Resuspend pellet in 30uL TE buffer.
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*Use caution when using phenol.  If you get any on you wash immediately with lots of water, it will turn your skin white. Eyeprotection is suggested.
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<h3>Telt Buffer (Usually in 1.5 mL aliquots in -20 restriction enzyme freezer)</h3>
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Final Concentrations
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*50mM Tris, pH 8.0
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*62.5mM EDTA, pH 8
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*2.5MLiCl
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*4% Triton X-100
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What you need to add:
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*5mL 1M Tris, pH 8.0
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*12.5mL 500mM EDTA, pH 8
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*31.5mL 8 M LiCl
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*4mL Triton (nanopure)
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Do Not Sterilize (autoclave or filger)
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<h3>DO NOT put pipettes into stock bottles, pour some into a tube for your own use.</h3>
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Phenol/chloroform is in a freezer pre-made
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70% ethanol is made with 700mL 100% ethanol, 300mL nanopure water.
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TE buffer is in sterile DNA solutions cabinet if there is none-make up with the 100X TE stock and nanopure water, autclave in 100mL aliquotes in dilution bottles.
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Procedure is from :
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Ausubel et al., current protocols in molecular biology.

Current revision

Telt Method -DNA Plasmid Preperation

note: This method is "not very good" in that it does not seperate plasmid DNA from chromosomal DNA. The advantage of this is that if there is no plasmid, you will get the chromosomal DNA. Use this prep to seperate the chromosomal DNA when there is no plasmid in the cell.

  • Use sterile techniques throughout
  • all centrifuge steps are at room temperature
  • put a tube of 100% ethanol on ice at start of procedure
  1. Grow cells overnight in 3mL LB (with appropriate antibiotics and sugars) in a 13 x 100mm tube at 37C (or other appropriate temperature) in a rotator.
  2. Pour 1.5 mL into sterile microcentrifuge tube. Centrifuge cells for 30 sec. Remove supernatent by aspiration
  3. Resuspend cells in 100uL TELT buffer. Vortex to resuspend pellet.
  4. Add 100uL 1:1 Phenol/Chloroform.
  5. Vortex for 15 sec. (Can leave cells for 5 minutes on th ebench).
  6. Centfigue for 1min. Transfer top layer to clean tube.
  7. Add 100uL chloroform/isoamyl (24:1)
  8. Vortex for 15 sec. (Can leave cells for 5 minutes on th ebench.)
  9. Centrifuge for 1 min.
  10. Transfer 75uL of the supernatent to a new microcenttrifuge tube.
  11. Add 200uL of 100% EtOH (cold). Mix well by inversion. Incubate at room temperature 2-10 minutes.
  12. Centifuge for 10 minutes.
  13. Pour off supernatant. Add 1mL 70% EtOH. Invert several times. Pour off liquid. Spin briefly to bring liquid to bottom of tube. Remove rest of liquid by aspiration, being careful not to disturb pellet.
  14. Air dry pellet (about 1/2 hour). or vacuum dry 5 minutes.
  15. Resuspend pellet in 30uL TE buffer.
  • Use caution when using phenol. If you get any on you wash immediately with lots of water, it will turn your skin white. Eyeprotection is suggested.

Telt Buffer (Usually in 1.5 mL aliquots in -20 restriction enzyme freezer)

Final Concentrations

  • 50mM Tris, pH 8.0
  • 62.5mM EDTA, pH 8
  • 2.5MLiCl
  • 4% Triton X-100

What you need to add:

  • 5mL 1M Tris, pH 8.0
  • 12.5mL 500mM EDTA, pH 8
  • 31.5mL 8 M LiCl
  • 4mL Triton (nanopure)

Do Not Sterilize (autoclave or filger)

DO NOT put pipettes into stock bottles, pour some into a tube for your own use.

Phenol/chloroform is in a freezer pre-made

70% ethanol is made with 700mL 100% ethanol, 300mL nanopure water.

TE buffer is in sterile DNA solutions cabinet if there is none-make up with the 100X TE stock and nanopure water, autclave in 100mL aliquotes in dilution bottles.

Procedure is from : Ausubel et al., current protocols in molecular biology.

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