Team:UNICAMP-EMSE Brazil/protocols/Mini prep

Miniprep:

• After bacterial growth in liquid media, in sterile conditions, aliquotate 1 mL of the culture in an eppendorf tube with final concentration of 30% glycerol (remember to check it´s stock concentration to calculate). Gently mix it and store at -20 °C.
• With the other 3 mL:

1. Pour 1.5 ml of the culture into a microfuge tube. Centrifuge at maximum speed for 30 seconds at 4°C in a microfuge. Repeat this operation with the other 1,5 mL.
2. Remove the medium by aspiration (using the pipette), leaving the bacterial pellet as dry as possible.
3. Resuspend the bacterial pellet in 100 μl of ice-cold Alkaline lysis solution I by vigorous vortexing.
4. Add 200 μl of freshly prepared Alkaline lysis solution II to each bacterial suspension. Close the tube tightly, and mix the contents by inverting the tube rapidly five times. Do not vortex! Store the tube on ice.
5. Add 150 μl of ice-cold Alkaline lysis solution III. Close the tube and disperse Alkaline lysis solution III through the viscous bacterial lysate by inverting the tube several times. Store the tube on ice for 3-5 minutes.
6. Centrifuge the bacterial lysate at maximum speed for 5 minutes at 4°C in a microfuge. Transfer the supernatant to a fresh tube.

• (Optional) Add an equal volume of phenol:chloroform. Mix the organic and aqueous phases by vortexing and then centrifuge the emulsion at maximum speed for 2 minutes at 4°C in a microfuge. Transfer the aqueous upper layer to a fresh tube.

1. Precipitate nucleic acids from the supernatant by adding 0,7 volumes of isopropanol at room temperature. Mix the solution by vortexing and then allow the mixture to stand for 2 minutes at room temperature.
2. Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 minutes at 4°C in a microfuge.
3. Remove the supernatant by gentle aspiration. Stand the tube in an inverted position on a paper towel to allow all of the fluid to drain away. Use a Kimwipe or disposable pipette tip to remove any drops of fluid adhering to the walls of the tube.
4. Add 1 ml of 70% ethanol to the pellet and invert the closed tube several times. Recover the DNA by centrifugation at maximum speed for 2 minutes at 4°C in a microfuge.
5. Remove all of the supernatant by gentle aspiration. Take care with this step, as the pellet sometimes does not adhere tightly to the tube.
6. Remove any beads of ethanol that form on the sides of the tube. Store the open tube at room temperature until the ethanol has evaporated and no fluid is visible in the tube (5-10 minutes).
7. Dissolve the nucleic acids in 50 μl of milliQ water. Vortex the solution gently for a few seconds. Store the DNA solution at -20°C.
8. Quantificate the plasmid using absorbance at 260 nm (One absorbance unit at 260 nm corresponds to 50 μg/mL of DNA) and 280 nm.
9. Concentration of plasmid (μg/mL) = A260*50*dilution
10. Ratio 260/280 nm should be between 1,8-2,0.

RECIPES

• Alkaline Lysis Solution I

• 50 mM glucose
• 25 mM Tris-Cl (pH 8.0)
• 10 mM EDTA (pH 8.0)
• Prepare Solution I from standard stocks in batches of approx. 100 ml, filtrate in 0,22 μm, and store at 4°C.

• Alkaline Lysis Solution II

• 0.2 N NaOH (freshly diluted from a 10 N stock)
• 1% (w/v) SDS
• Prepare Solution II fresh and use at room temperature.

• Alkaline Lysis Solution III

• 5 M potassium acetate, 60.0 ml
• glacial acetic acid, 11.5 ml
• H2O, 28.5 ml
• The resulting solution is 3 M with respect to potassium and 5 M with respect to acetate. Store the solution at 4°C and transfer it to an ice bucket just before use.

• EDTA

• To prepare EDTA at 0.5 M (pH 8.0): Add 186.1 g of disodium EDTA•2H2O to 800 ml of H2O. Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (approx. 20 g of NaOH pellets). Dispense into aliquots and sterilize by autoclaving. The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to approx. 8.0 by the addition of NaOH.

• Glycerol

• To prepare a 10% (v/v) solution: Dilute 1 volume of molecular-biologygrade glycerol in 9 volumes of sterile pure H2O. Sterilize the solution by passing it through a prerinsed 0.22-μm filter. Store in 200-ml aliquots at 4°C.

Miniprep using Kits:

MiniPrep (Fermentas GENEJET Plasid Miniprep kit - adapted)

1. Pellet the cells cetrifuging for 10 minutes at maximum speed.

2. Resuspend the pelleted cells in 250 μl of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.

3. Add 250 μl of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.

4. Add 350 μl of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times.

5. Centrifuge for 5 min to pellet cell debris and chromosomal DNA.

6. Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting.

7. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.

8. Add 500 μl of the Wash Solution to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.

9. Repet step 8.

10. Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution.

11. Transfer the GeneJET spin column into a fresh 1.5 ml microcentrifuge tube. Add 50 μl of pre-heated (60˚C) Elution Buffer or sterile water to the center of GeneJET spin column membrane.Incubate for 10 min at room temperature and centrifuge for 2 min.

12. Store the purified plasmid at -20˚C.