Team:Debrecen-Hungary/protocols/midiprep

Scientific Background

Overview

Procedure for High Copy Number Plasmids

Materials

1. Sample material: 30 ml E. coli culture, transformed with a high copy number plasmid Harvest cultures at a density between 2.0 and 6.0 A600 units per ml bacterial culture

2. Media: The isolation method is optimized for cultures grown in LB media; other rich media may require increased volumes of Suspension-, Lysis- and Neutralization Buffer, and an additional wash step

3. Plasmid size: The isolation procedure is suitable for all plasmid sizes; lysates of larger constructs (up to 100 kb) should be cleared by filtration to avoid shearing

4. Suspension Buffer/RNase A: To dissolve the lyophilized enzyme in Suspension Buffer, pipet 1 ml of Suspension Buffer (bottle 1, black cap) into the glass vial containing the lyophilized RNase (bottle 2, black cap). Reinsert the rubber stopper and invert the vial until all lyophilizate (including any that might stick to the rubber stopper) is dissolved. Transfer the dissolved enzyme back to the Suspension Buffer bottle (bottle 1). This is enough working solution for 60 Midi preps (isolation of up to 100 μg plasmid DNA/preparation)

5. If preparing aliquots of the working solution, remember that the final concentration of RNase A in the working solution must be 100 g/ml. Reconstituted buffer is stable for 6 months is stored properly (+2 to +8°C)

6. Neutralization Buffer: Before starting, cool it down to 4°C

7. Elution Buffer: Before starting, warm up the buffer to 50°C

8. Ethanol: Use 70% ethanol. Before starting, cool it down to 4°C

Procedure

1. Centrifuge bacterial cells from 30 ml culture grown in LB medium by centrifuging for 10 min at 5000 rpm,4°C
Discard the supernatant
Carefully resuspend the pellet in 4 ml Suspension Buffer + RNase and mix well

2. Add 4 ml Lysis Buffer to the suspension and mix gently by inverting the tube 6 times
Incubate 2-3 min at room temperature
To avoid shearing genomic DNA, do not vortex the suspension in Lysis Buffer. To prevent release of chromosomal DNA from the cell debris, do not incubate for more than 5 minutes

3. Add 4 ml chilled Neutralization Buffer to the suspension

Immediately mix the suspension gently by inverting the tube 6 times until a homogenous suspension is formed

Incubate the tube 5 min on ice

The solution becomes cloudy and a flocculent precipitate will form

4. Clear the lysate by filtration

Put a folded filter into a funnel that has been inserted into a 50 ml plastic tube

Moisten the filter with a few drops of Equilibrium Buffer

Load the lysate onto the wet, folded filter and collect the flowthrough The SDS precipitates with cellular debris when Neutralisation Buffer is added; this white precipitate should not be loaded onto the column. If the solution obtained after step 14 is not clear, remove the remaining precipitate by passing the solution over a folded filter

5. Mount the sealing ring to the column to fix the column in the collection tube

Insert one column into one collection tube

Equilibrate the column with 2,5 ml Equilibration Buffer

Allow the column to empty by gravity flow

Discard the flowthrough

6. Load the cleared lysate from step 4 onto the equilibrate column

Allow the column to empty by gravity flow

Discard the flowthrough

7. Wash the column with 5 ml Wash Buffer

Allow the column to empty by gravity flow

Discard the flowthrough

8. Repeat step 7

Discard flowthrough and collection tube

9. Re-insert the column into a new collection tube

Elute the plasmid with 5 ml prewarmed Elution Buffer (50°C)

Allow the column to empty by gravity flow

The collection flowthrough contains the plasmid

Elute the plasmid again with the flowthrough

Allow the column to empty by gravity flow

Plasmid concentration is higher, than after only one elution

10. Precipitate the eluted plasmid DNA with 3,6 ml isopropanol

Total volume ~6,8 ml

Divide the eluted plasmid into five 1,5 ml eppendorf tubes and one tube which contains 73 μl.

Centrifuge immediately 30 min at 15000×g (rcf), +4°C.

Carefully discard the supernatant.

11. Divide the 6 tubes into two groups.

Wash the plasmid DNA with 1,5 ml chilled (+4°C) 70% ethanol in the first tube and afterwards wash the other two with the same ethanol.

Wash the other group (3 tubes) the same way.

Centrifuge 10 min at 15000×g (rcf), +4°C.

Carefully remove ethanol from the tube with pipet tip.

Air-dry the plasmid DNA pellet for 10 min.

12. Carefully redissolve the plasmid DNA pellet in 100 μl TE buffer.

13. Measure the concentration with NanoDrop (DNA)

Notes & troubleshooting

References

1.Birnboim, H.C. and Doly, J. (1979) A rapid alkaline lysis procedure for screening recombinant plasmid DNA. Nucl. Acids Res. 7, 1513-1522

2.Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd. Edition, Cold Spring Harbour Laboratory Press

3.Ausubel, F.M. et al. (eds.) (1991) Current Protocols in Molecular Biology, Wiley Interscience, New York

Other