Team:Hawaii/Protocols/Plasmid prep

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 &mu;l 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 &mu;l 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 &mu;l 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 &mu;l TE buffer.

Reference: Short Protocols in Molecular Biology


 * Add 1 &mu;l RNase after step 12. Incubate at 55C for 30-90 min. (Re: SC)
 * Add 1 &mu;l 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).
 * 3) *use baffled 2L flask
 * 4) *for this prep, we used a 300mL culture.
 * 5) Centrifuge 10 minutes at maximum 894 rcf (maximum rcf for our centrifuge), 4&deg;C. Use 50mL aliquots.
 * 6) *The protocol recommends using 6,000 x g.
 * 7) *The 300mL prep is divided into 6 50mL tubes.
 * 8) Combine 3 tubes by resuspending in 4mL of GTE solution. Incubate 10 minutes at room temperature.
 * 9) Add 10mL NaOH/SDS solution, mix (gently) by inverting 4 times. Incubate on ice for 10 minutes.
 * 10) *Solution should become cloudy because cells have lysed.
 * 11) * Add RNase at a concentration of 50 &mu;g/ml after this step if desired.
 * 12) Add 7.5mL potassium acetate, mix (very gently) by inverting (slowly) 4 times. Incubate on ice for 10 minutes.
 * 13) *A white precipitate forms.
 * 14) * Incubating on ice longer (i.e. "freezing" proteins) will help pellet the proteins better
 * 15) Centrifuge for 15 minutes at 894 rcf, 4&deg;C.
 * 16) *Recommended to spin at 20,000 x g for 10 minutes.
 * 17) *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.
 * 18) Decant supernatant to a new tube.
 * 19) *Do this step with a pipette and avoid the white precipitate.
 * 20) Add 0.6 volume of isopropanol. Mix by inversion, let stand 5-10 minutes at room temperature.
 * 21) *For a 21.5mL prep (total volume up to this point) add 12.9mL isopropanol.
 * 22) Centrifuge 15 minutes at max rcf (894 rcf for us) at room temperature. Discard supernatant.
 * 23) *Recommended to spin at 15,000 x g.
 * 24) *Centrifuge until really good pellet forms. Avoid the pellet!
 * 25) Wash pellet with 2mL 70% ethanol.
 * 26) Centrifuge for 5 minutes at 894 rcf at room temperature. Aspirate ethanol.
 * 27) *Recommended to spin at 15,000 x g briefly.
 * 28) *Centrifuge until really good pellet forms.
 * 29) Dry pellet in the hood.
 * 30) * In a really good plasmid prep, the pellet should be clear (free of protein).
 * 31) *Recommended to dry the pellet under vacuum.
 * 32) Resuspend in 100uL TE, lightly vortex.
 * 33) *Recommended to store indefinitely at 4&deg;C (but does not specify if buffer is needed).
 * 34) * Resuspend in a smaller volume of TE for higher concentration of plasmid.
 * 35) * If solution is viscous (like egg whites), there is a lot of protein contamination.
 * 36) Heat at 65&deg;C for 30 minutes.
 * 37) *The product was cloudy so taking extra purification step.
 * 38) Centrifuge 10 minutes at 894 rcf, room temperature.
 * 39) Aspirate clear liquid, avoiding pellet.
 * 40) Wash pellet with 100uL TE, centrifuge, aspirate and combine with product.
 * 41) Check the concentration of the plasmid using a spectrophotometer (need protcol).
 * 42) 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.