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Contents 1 Large-Scale Preparation of Plasmid DNA 1.1 Methods 2 Large-Scale Plasmid Prep Procedure 3 UV Spectroscopy for the Quantification of Plasmid DNA 4 Restriction Digest and Gel Electrophoresis for Verification and Quantification of pRL1383a 5 Procedure for a 10uL Digestion/Gel Electrophoresis 6 UV Spectrometric Quantification 6.1 Electrophoresis Results 6.2 Discussion

Large-Scale Preparation of Plasmid DNA

A protocol for the preparation of plasmid DNA from large cultures. Adapted from a protocol for a 500mL prep. Yields milligram quantities of reasonably clean crude DNA. To obtain highly purified plasmid DNA can be obtained by using CsCl/ethidium bromide equilibrium centrifugation (protocol found in Maniatis).

Also included are two assays for verification and quantification of plasmid DNA: UV wpectrometric quantification and restriction digest followed by gel electrophoresis.

Methods

Large-Scale Plasmid Prep Procedure

Materials

• LB medium containing selective agent
• Plasmid bearing E. coli strain
• GTE solution (50mM Glucose/10mM EDTA/25mM Tris-HCl, pH 8.0)
• NaOH/SDS solution (0.2M NaOH/1.0%SDS)
• 10mg/mL Rnase in Tris-HCl, pH 7.5
• Isopropanol
• 70% (v/v) ethanol
• centrifuge
• 2L baffled flask
• 50mL centrifuge tubes
• Water Bath at 55°C

1. Innoculate 5mL LB containing selective agent with a colony of plasmid bearing E. coli. Grow at 37C with vigorous shaking over night.
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.
   • For one variation (latter called Prep 1), add 50ug/mL Rnase solution (20uL).
5. Add 10mL NaOH/SDS solution, mix (gently) by inverting 4 times. Incubate on ice for 10 minutes.
   • Solution should become homogeneous and clear. This prep was not clear, but proceeded with protocol.
6. Add 7.5mL potassium acetate, mix (gently)by inverting 4 times. Incubate on ice for 10 minutes.
   • A white precipitate forms.
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. 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.
   • For one variation (latter called Prep 2), add 50ug/mL Rnase solution (20uL).
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 894 rcf 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.
    • 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).
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

Restriction Digest and Gel Electrophoresis for Verification and Quantification of pRL1383a

Procedure for a 10uL Digestion/Gel Electrophoresis

Materials

• EcoRI
• EcoRI buffer
• BSA
• nanopure water
• pRL1383a (from prep 1 and prep 2)
• heat block at 37°C (later 65°C)
• 1.5 mL centrifuge tubes
• agarose gel
• gel apparatus
• Ethidium Bromide
• 6X loading buffer
• Ladder

1. Turn on heat block.
2. To the centrifuge tube, add 2uL nanopure water, 1uL EcoRI buffer, 1uL BSA, 5uL pRL1383a plasmid prep.
3. Microcentrifuge until all contents are in solution at bottom of tube.
4. Add 1uL EcoRI.
5. Place on heat block for 1 hour.
6. While waiting, prepare the gel.
   1. Microwave the gel in bottle in 30 second intervals, mixing in between until all of gel is melted.
   2. add Ethidium Bromide to gel.
   • There should be some already in the gel, adjust as needed.
   1. Put in well clip, pour gel after it is sufficiently cooled (does not hurt when you touch it).
7. After 1 hour, increase heat on block to 65°C and incubate digestion for 20 minutes.
   • Heat inactivation of EcoRI.
8. Prepare samples for gel electrophoresis:
   1. Plan out wells, in addition to digestion, add circular plasmid as a control.
   2. Mix sample + loading buffer in 5:1 ratio.
9. Load Ladder (need name of ladder) and samples.
10. Run gel at 95V for 45 minutes or until end of ladder reaches halfway down gel.
11. Take some pictures.

UV Spectrometric Quantification

Electrophoresis Results

Electrophoresis results of Circular DNA

Lane	Contents	Description
Lane 1	The ladder	Faint, but sufficiently separated
Lane 2	Prep 1, no dilution	Most of the DNA appears to have remained in the well, a smear indicates some has run to about 10kb.
Lane 3	Prep 2, no dilution	Most of the DNA appears to have remained in the well, a faint smear indicates some has run to about 10kb.
Lane 4	Prep 1, 1:10 dilution	DNA appears to have remained in the well.
Lane 5	Prep 2, 1:10 dilution	DNA appears to have remained in the well.
Lane 6	Prep 1, 1:100 dilution.	No nucleic acids stained.
Lane 7	Prep 2, 1:100 dilution	No nucleic acids stained.

Electrophoresis results of Linear DNA

Lane	Contents	Description
Lane 1	The ladder	Sufficiently separated
Lane 2	Prep 1 Circular DNA (Control)	One bright band in high bp region, one bright band near 10kb region, large smear in low kb region.
Lane 3	Prep 1, Linear DNA	No nucleic acids stained.
Lane 4	Prep 2 Circular DNA (Control)	One semi-bright band in high bp region, one bright band near 10kb region, small smear in low kb region.
Lane 5	Prep 2, Linear DNA	One bright band in high bp region, one bright band near 10kb region, small smear in low kb region.

Discussion

• A large scale plasmid prep can be performed with this procedure.
• From the gel electrophoresis results, is not apparent that the step in which the Rnase is added is important because a smear in the low kb region is found in both preps indicating a possible RNA contamination. We should add as the Maniatis manual says after the GTE addition so that the experiment can follow this manual as closely as possible.
• To improve the RNA digestion, we will adjust the protocol by incubating the RNase for 30 minutes at 55°C.
• From the gel electrophoresis results, we can conclude that it is necessary to linearize the plasmid DNA before a gel is run. It is also apparent that levels of Ethidium Bromide should be added so that sufficient staining is achieved.
• This protocol needs to be updated with the UV Spec quantification experiment and the concentrations calculated (they are locked in the lab right now!).

Insanity is doing the same thing over and over again and expecting different results. - Albert Einstein

[http://manoa.hawaii.edu/ ][http://manoa.hawaii.edu/ovcrge/ ][http://www.ctahr.hawaii.edu ]

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