Streptomyces:Protocols／MiniMaxiPrep

Small Scale Plasmid Isolation (Mini / Maxi Prep)

Description 
A mini prep / maxi prep is used to isolate plasmid or cosmid DNA from bacteria, normally E.coli. An overnight culture of cells is lysed and the majority of the cells contents are removed with the DNA remaining dissolved in solution. Phenol/Chloroform extraction is used to purify the DNA contaminated by histones and other soluble proteins. Equal volumes of a phenol:chloroform mixture and the aqueous DNA sample are mixed, forming a biphasic mixture. The proteins partition into the organic phase while the DNA (as well as other contaminants such as salts, sugars, etc.) remain in the aqueous phase. This (top) phase is removed and the DNA precipitated with alcohol; all anionic salts remain in the aqueous liquid. Washing with 70% ethanol removes the last of the contaminants leaving the final product.

Uses 
Extract large quantities of good quality plasmid DNA for further work, e.g. sub-cloning, transformations, verification by digestion/sequencing, PCR, southern blots etc…

Prerequisites 
An overnight culture needs to be grown of the bacterial strain containing the required plasmid/cosmid to be extracted. A fume cupboard dedicated to phenol/chloroform work & waste needs to be available.

Safety 
COSHH forms need to be created for the lab in which the work is done. All users need to understand the risks associated with both phenol & chloroform. A phenol decontamination kit (PEG300:Meths) must be located near the fume hood in which the work is done. General laboratory & molecular microbiology safety rules apply.

Requirements 
Cells, ice, 15mL falcon tube(s), micro-centrifuge tube(s) (MCT), Solution I, Solution II, Solution III, Phenol, Chloroform, isopropanol (propan-2-ol), 70% ethanol, sdH₂O (sterile distilled water), falcon centrifuge @ ~4°C, micro-centrifuge @ ~4°C. 
Optional: incubator (i.e. a water bath) @ 37°C, Ribonuclease A (RNase).

Preparation 
Chill Solution I and Solution III on ice; the isopropanol and 70% ethanol should be placed in the freezer. Make fresh Solution II (see notes). Label the falcons or MCTs. Ensure the correct sized centrifuge is set to ~4°C.

Method 
As the cells will be lysed and DNA extracted there is no requirement for sterility. All steps should be performed chilled (on ice) except for the phenol/chloroform step(s) and the RNase incubation step (obviously!). Spins should be done at the maximum capacity of the centrifuge/tubes. A mini prep uses 3mL of cells; a maxi prep uses 50mL of cells (different steps are indicated in bold). 

Cell lysis:

1. Take the overnight culture and spin down 1.5mL (50mL) of cells for 3 (5) minutes.

2. Pour off the supernatant and add another 1.5mL of cells, spin.

3. Re-suspend the pellet in 100μL (1mL) of ice cold Solution I.

4. Add 200μL (2mL) of Solution II and gently invert the tube (roll the falcon) to mix. Leave on ice for 5 minutes.

5. Add 150μL (1.5mL) of Solution III and shake vigorously. Leave on ice for 10 minutes.

6. Centrifuge for 5 minutes.

Phenol/Chloroform Cleanup:

1. Transfer the supernatant by pipetting to a clean MCT (15mL falcon).

2. Add 0.5 volumes of phenol and 0.5 volumes of chloroform to 1 volume of supernatant ~250:250->500μL (2.5:2.5->5mL).

3. Mix well by vortexing and centrifuge for 5 minutes.

4. Carefully take the top aqueous phase and transfer to a new tube.

RNase (optional):

1. Add RNase to a final concentration of 3μg/mL (5μL of 3mg/mL diluted stock / 5μL of 30mg/mL stock).

2. Incubate in a 37°C water bath for 30-45 minutes.

3. Repeat the phenol/chloroform step.

Alcohol Precipitation / Purification:

1. Add an equal volume of ice cold isopropanol to the aqueous solution.

2. Mix well by inversion and leave on ice for 10 minutes.

3. Centrifuge at maximum speed for 15 minutes.

4. Carefully pour off the isopropanol leaving the DNA pellet.

5. Wash the pellet with 200μL (2mL) of 70% ethanol by pipetting.

6. Centrifuge for 5 minutes.

7. Remove all the alcohol by pipetting and air dry the pellet for up to 30 minutes.

8. Re-suspend the DNA pellet in 30-50μL (200-300μL) sdH₂O.

9. Store the samples at -20°C.

Notes 
Solution I: 50mM Tris?HCl pH8.0 / 10mM EDTA pH8.0 
Solution II: 200mM NaOH / 1% SDS 
Solution III: 3M KOAc (Potassium Acetate) pH5.5

After re-suspending the cells in Solution I the mixture should be homogenous. The addition of Solution II causes a translucent goo of lysed cell matter to be observed. Solution III precipitates the majority of this by instant decrease in pH. 
Solution II contains sodium hydroxide which is critical in lysing the cells. Ideally it needs to be made fresh or at least from a fresh solution of sodium hydroxide. This is because the sodium hydroxide absorbs carbon dioxide from the atmosphere causing it to change into sodium carbonate, which will not lyse the cells. After the addition of Solution II gentle mixing is required so the DNA is not damaged. The DNA will have various (relatively large) proteins bound and anything more than gentle inversion or rolling the falcon can shear the DNA. 
Phenol is toxic and should be handled with extreme care. Its solution is buffered with 3% NaCl, which forms an upper biphasic layer. When using the phenol ensure you pipette from below this salt buffer layer. When removing the top aqueous phase after treating with phenol/chloroform be careful not to disturb the lower organic layer. Any phenol taken with the aqueous phase may degrade the RNase or affect later reactions, such as PCR. 
If the alcohol solutions are ice cold and the samples centrifuged @ ~4°C the DNA pellet will become more visible and therefore, easier to work with. Also, the yield may be slightly higher. 
The addition of RNase is a worthwhile step, especially for maxi preps. It makes the analysis of future reactions easier, for example, the viewing of small fragments after a restriction digest.