Team:Hawaii/Protocols/Plasmid prep

From 2008.igem.org

Contents

Miniprep Protocol

1. Grow single colony of E. coli at 37C overnight in 5 ml LB w/ antibiotic selection.
2. Microcentrifuge 1.5 ml cells for 20 sec at 16,000g. Discard supernatant.
3. Resuspend pellet in 100 μl GTE solution.

  • 50 mM glucose
  • 10 mM EDTA
  • 25 mM Tris-HCl (pH 8.0)

4. Let sit for 5 min. at room temperature.
5. Add 200 μl NaOH/SDS solution.

  • 0.2 M NaOH
  • 1% SDS

6. Mix by inverting the tube a few times.
7. Incubate on ice for 5 min.

  • Incubate no more than 5 min. to allow for maximum release of plasmid DNA while minimizing genomic DNA release and overexposure to denaturing conditions.

8. Add 150 μl potassium acetate solution.

  • Precipitation of cellular debris may be enhanced by using chilled KOAc.

9. Invert a few times to mix.
10. Incubate on ice for 5 min.
11. Microcentrifuge for 3 min. at 16,000g.
12. Transfer supernatant to a new tube.
13. Add 0.8 ml 95% ethanol.
14. Incubate for 2 min. at room temperature.
15. Microcentrifuge for 1 min. at 16,000g at room temperature. Remove supernatant.
16. Wash pellet w/ 1 ml 70% ethanol. Aspirate to dry (dry in hood).
17. Resuspend pellet in 30 μl TE buffer.

Reference: Short Protocols in Molecular Biology

  • Add 1 μl RNase after step 12. Incubate at 55C for 30-90 min. (Re: SC)
  • Add 1 μl RNase after step 7. Incubate at 55C for 60 min. -GK

Large Scale Prep

  1. Innoculate 5mL LB containing selective agent with a colony of plasmid bearing E. coli. Grow at 37C with vigorous shaking (~235 rpm) overnight.
  2. Inoculate 500mL LB containing selective agent with ~1mL of over night culture. Grow at 37C with vigorous shaking until OD600~4.0 is reached (saturation).
    • use baffled 2L flask
    • for this prep, we used a 300mL culture.
  3. Centrifuge 10 minutes at maximum 894 rcf (maximum rcf for our centrifuge), 4°C. Use 50mL aliquots.
    • The protocol recommends using 6,000 x g.
    • The 300mL prep is divided into 6 50mL tubes.
  4. Combine 3 tubes by resuspending in 4mL of GTE solution. Incubate 10 minutes at room temperature.
  5. Add 10mL NaOH/SDS solution, mix (gently) by inverting 4 times. Incubate on ice for 10 minutes.
    • Solution should become cloudy because cells have lysed.
    • Add RNase at a concentration of 50 μg/ml after this step if desired.
  6. Add 7.5mL potassium acetate, mix (very gently) by inverting (slowly) 4 times. Incubate on ice for 10 minutes.
    • A white precipitate forms.
    • Incubating on ice longer (i.e. "freezing" proteins) will help pellet the proteins better
  7. Centrifuge for 15 minutes at 894 rcf, 4°C.
    • Recommended to spin at 20,000 x g for 10 minutes.
    • Centrifuge until a good pellet forms (may take up to 90 min). Some material will be floating, remove as much as possible with pipette tip.
  8. Decant supernatant to a new tube.
    • Do this step with a pipette and avoid the white precipitate.
  9. Add 0.6 volume of isopropanol. Mix by inversion, let stand 5-10 minutes at room temperature.
    • For a 21.5mL prep (total volume up to this point) add 12.9mL isopropanol.
  10. Centrifuge 15 minutes at max rcf (894 rcf for us) at room temperature. Discard supernatant.
    • Recommended to spin at 15,000 x g.
    • Centrifuge until really good pellet forms. Avoid the pellet!
  11. Wash pellet with 2mL 70% ethanol.
  12. Centrifuge for 5 minutes at 894 rcf at room temperature. Aspirate ethanol.
    • Recommended to spin at 15,000 x g briefly.
    • Centrifuge until really good pellet forms.
  13. Dry pellet in the hood.
    • In a really good plasmid prep, the pellet should be clear (free of protein).
    • Recommended to dry the pellet under vacuum.
  14. Resuspend in 100uL TE, lightly vortex.
    • Recommended to store indefinitely at 4°C (but does not specify if buffer is needed).
    • Resuspend in a smaller volume of TE for higher concentration of plasmid.
    • If solution is viscous (like egg whites), there is a lot of protein contamination.
  15. Heat at 65°C for 30 minutes.
    • The product was cloudy so taking extra purification step.
  16. Centrifuge 10 minutes at 894 rcf, room temperature.
  17. Aspirate clear liquid, avoiding pellet.
  18. Wash pellet with 100uL TE, centrifuge, aspirate and combine with product.
  19. Check the concentration of the plasmid using a spectrophotometer (need protcol).
  20. Verify presence of plasmid DNA by first using a restriction digest, followed by gel electrophoresis.

UV Spectroscopy for the Quantification of Plasmid DNA

  • used to asses purity and concentration of nucleic acids
  • A260 measurements are quantitative for relatively pure nucleic acid preps in microgram quantities
  • Cannot be used to discriminate between RNA and DNA
  • Ratio of A260/A280 indicates purity, as protein absorbs at 280nm.
  • A325 indicates particulates in solution or dirty cuvette
  • A230 for contaminants containing peptide bonds or aromatic moieties such as protein and phenol

NanoDrop Protocol

  1. Turn on computer, select spec icon, choose nucleic acids setting
  2. Pull up lever of spec (DON'T PULL with WIRE!), wash top and bottom with small amount of water.
  3. Blank with 2uL TE, click blank
  4. Load 2uL sample, click measure
  5. If results significant, can print. If not, repeat steps with a dilution of sample.
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