Laboratory Protocols

Plasmid DNA isolation

For this practical, a strain of E. coli with a GFP-gene on its plasmid is required. This can be, for instance, the pGLO-plasmid, available in the Bio-Rad kit. This is a starter kit, which also contains all kinds of disposables and media. The plasmid pGFP-uv or another plasmid is also appropriate and may be available somewhere in your own institute or a nearby institute. Growth of a culture of the E. coli with the appropriate plasmid is prerequisite, and a protocol is given below. This is followed by protocols for isolation of the plasmid DNA from the E. coli in two different ways.

Prerequisite: Growing Bacteria in Liquid Media

Materials

1. LB medium: 10 g bacto tryptone and 5 g bacto yeast extract in 10 g NaCl are dissolved in 900!ml of demineralised water. After correcting the pH to 7.0, and adjusting the volume to 1000 ml, the medium is sterilised for 20 minutes at 121 C. After sterilisation and cooling down the medium (to below 50 C), ampicillin (stock solution: 50 mg / ml) is added to the medium with a final concentration of 80 g ampicillin per ml medium. 2. E. coli strain with the plasmid pGFPuv or pGLO for the production of GFP protein.
Procedure

1. Inoculate 20 ml LB medium with the E. coli pGFPuv or pGLO strain approximately 16 hours before the start of the isolation of plasmid DNA. 2. The culture is cultivated at 37 C under aeration with air (by rotating the vessels with medium at 200 rpm). 3. Before starting with the plasmid DNA isolation, the culture (with bacteria; how can you see if there has been bacterial growth?) is cooled in ice/water bath for 15 minutes. 4. Take from the culture 200 l and dilute this with 1800 l fresh LB medium. Measure the optical density at 660 nm (use LB medium as a blank). Extinction = ; dilution factor = Calculate the amount of cells in cells/ml (Extinction 660 nm = 1 corresponds to 10 9 cells/ml).

Laboratory Protocols
A. QIAGEN PROTOCOL
DNA Isolation Purpose

To isolate plasmid DNA on a small-scale - 1 to 20 g plasmid DNA - two methods will be referred to: that with the use of the Qiagen prep kit, and the alkaline lysis method.

The plasmid might be isolated from E. coli cells using a Qiagen Qiaprep Miniprep kit. The following procedure has been adapted from the Qiagen Protocol.
The QIAprep Principle

The QIAprep miniprep procedure is based on alkaline lysis of bacterial cells followed by adsorption of DNA onto silica in the presence of high salt. The unique silica-gel membrane used in QIAprep Miniprep Kits completely replaces glass or silica slurries for plasmid minipreps. The procedure consists of three basic steps: Preparation and clearing of a bacterial lysate Adsorption of DNA onto the QIAprep membrane Washing and elution of plasmid DNA All steps are performed without the use of phenol, chloroform, CsCI, ethidium bromide, and without alcohol precipitation.
Alkaline lysis of bacteria

The QIA-prep miniprep procedure uses the modified alkaline lysis method of Birnboim & Doly. Bacteria are lysated under alkaline conditions, and the lysate is subsequently neutralised and adjusted to high-salt binding conditions in one step, ready for purification on the QIA-prep silica-gel membrane. For more details on growth of bacterial cultures and alkaline lysis, please refer to original QIA-prep handbook.
Lysate clearing

In the QIA-prep Spin the lysates are cleared by centrifugation.

DNA adsorption to the QIAprep membrane

QIA-prep columns, strips, and plates use a silica-gel membrane for selective adsorption of plasmid DNA in high-salt buffer and elution in low-salt buffer. The optimised buffers in the lysis procedure combined with the unique silica-gel membrane ensure that only DNA will be adsorbed, while RNA, cellular proteins, and metabolites are not retained on the membrane but are found in the flow-through; see figure below.

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Materials

1. 2. 3. 4. 5. 6. 7. 8. 9.

Disposals: sterile vials, sterile pipette tips Micro pipettes Micro centrifuges Buffer P1: 50 mM Tris-HCl, pH 8.0, 10 mM EDTA and 100 g RNase A per ml Buffer P2: 200 mM NaOH and 1 % SDS Buffer N3: guanidine HCl and HAc, pH 4.5 Buffer PB: with guanidine HCl and isopropanol Buffer PE: with ethanol Buffer EB: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA

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Method Notes / questions

1. The yield of plasmid DNA is much higher if the bacteria are totally re-suspended. 2. Antibiotics and medium additives, such as vitamins, are highly thermo-instable; these solutions must be filtered sterilised (0.2 m) and are stored in a freezer (- 40 C) as a stock solution (10 up to 1000 times concentrated) 3. The quality of water for solutions is utmost important; sterile MQ water is normally used. 4. What are the functions of Tris-HCl, EDTA, RNase A, NaOH, SDS, guanidine HCl, HAc, isopropanol and ethanol? 5. What is the function of the silica column? 6. Why is the DNA solution stored preferentially in buffer EB (see above) and not in sterile MQ water? 7. Control actions: - Buffer P1 must contain RNase, - Check Buffers P2 and N3 before use for salt precipitation. Redissolve any precipitate by warming to 37 C. Do not shake vigorously! - Buffer PE must contain ethanol 8. All centrifugation steps are carried out at maximum speed (13,000 rpm) in a conventional microcentrifuge. Buffers P2, N3 and PB contain irritants. Wear gloves when handling these buffers.
Using a microcentrifuge

This protocol is designed for purification of up to 20 g of high-copy plasmid DNA from 1 up to 5 ml overnight cultures of E. coli in LB (Luria-Bertani) medium. For purification of low-copy plasmids and cosmids, large plasmids (>10 kb), and DNA prepared using other methods, some modifications must be applied.
Procedure

1. Resuspend the pellet of bacterial cells in 250 l of Buffer P1 and transfer to a microfuge tube. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspending the pellet. 2. Add 250 l of Buffer P2 and gently invert the tube 4-6 times to mix. Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. lf necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 minutes. 3. Add 350 l of Buffer N3 and invert the tube immediately but gently 4-6 times. To avoid localised precipitation, mix the solution gently but thoroughly, immediately after addition of Buffer N3. The solution should become cloudy. 4. Centrifuge for 10 min. A compact white pellet will form. During centrifugation, place a QIA-prep spin column in a 2-ml collection tube. 5. Apply the supernatants from step 4 to the QIA-prep column by decanting or pipetting. 6. Centrifuge 30-60 sec. Discard the flow-through. 7. (Optional): Wash QIA-prep spin column by adding 0.5 ml of Buffer PB and centrifuging 30-60 sec. Discard the flow-through. This step is necessary to remove trace nuclease activity when using end-A + strains such as the E. coli JM series, HB 101 and its derivatives, or any wild-type E. coli strain,

Laboratory Protocols
which have high levels of nuclease activity or high carbohydrate content. Host strains such as XL-1 Blue and DH5! do not require this additional wash step. 8. Wash QIA-prep spin column by adding 0.75 ml of Buffer PE and centrifuging 30-60 sec. 9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer. IMPORTANT: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit elution from the column and subsequent enzymatic reactions. 10. Place QIA-prep column in a clean, sterile 1.5-ml microfuge tube. To elute DNA, add 50 l of Buffer EB (10 mM Tris-Cl, pH 8.5) or H2O to the centre of each QIA-prep column, let stand for 1 min, and centrifuge for 1 min. Repeat this step by adding again 50 l of Buffer EB (10 mM Tris-Cl, pH 8.5) or H2O to the centre of each QIA-prep column, let stand for 1 min, and centrifuge for 1 min. 11. Take from the obtained DNA solution (approximately 100 l) 20 l and add 1980 l sterile MQ water; so the dilution factor = . 12. Transfer this diluted DNA solution to a quartz optical cell and measure the optical density at 260 and 280 nm in a spectrum range from 200 up to 400 nm (see for more details: protocol 3: measurement of DNA and RNA) 13. The undiluted DNA solution should be coded and stored in a freezer.
Questions / tasks:

1. Calculate the DNA concentration of the diluted and undiluted DNA solutions. A DNA solution of 1 mg DNA / ml gives an optical density of 20.0 at 260 nm. 2. Calculate the yield of total DNA in g; so the undiluted solutions contains . g / l. 3. How many mls of cultivated bacteria are used for the DNA isolation; this means that, per ml cultivated bacteria, . g DNA is isolated. 4. After correction for the amount of bacteria cells per ml and the optical density of the broth you get a DNA yield of g per bacterium cell. Answers to notes/questions:
3. The function of the following solutions/chemicals: Tris-HCl: buffer solution EDTA: binds 2+ ions like Magnesium Rnase: breaks down the RNA in the cell NaOH: (temporarily) denatures DNA SDS: soap-like solution, dissolves membranes and denatures proteins Guanidine HCl: removes traces of nuclease activity HAc: precipitation of SDS-cell wall protein complexes, neutralisation of the (NaOH) solution Isopropanol: aggregates plasmid DNA Ethanol: wash step, removal of isopropanol and rests of salts 4. The function of the silica column is: 5. EB contains EDTA and Tris

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B. Alkaline lysis method
Notes / questions

1. Make a work schedule. Illustrate where your plasmid will be at each point in the procedure. The principle of the method is explained here; match this with the steps of the protocol. 2. TEG (Tris, EDTA glucose solution) with RNase breaks down the bacteria in isotonic environments. RNase specifically breaks down the RNA in the cell 3. SDS (sodium dodecyl sulphate, a soap-like solution) dissolves the membranes and denatures the proteins, NaOH (temporarily) denatures the DNA. 4. Ice cold 7.5 M ammonium acetate precipitates the SDS-protein and cell wall remains. This is also a neutralisation step, DNA can partially renature. Chromosomal DNA will not be able to renature easily (because of its large size) and will be removed by centrifugation. Plasmids are smaller, and will be renatured and stay in the supernatant. 5. The supernatant is transferred to a new Eppendorf tube, and isopropanol (isopropanol can be replaced by twice the volume of 96% ethanol) is added in order to let the plasmids aggregate. The washing with ethanol is meant to remove salts and remaining isopropanol. TE-buffer is the ideal solution to keep the DNA in, because the EDTA in it complexes magnesium ions that the enzyme DNase needs to work.
Material

250 ml 10 % SDS solution 100 ml 10 M NaOH 250 ml 70% ethanol 100 ml 7.5 M ammonium acetate pH 7.8 100 ml isopropanol 100 ml TE buffer pH 8.0 100 ml STE-buffer pH 8.0 50 ml o.n. (= over night) culture of your E. coli with the pGLO or pGFP-uv plasmid 100 ml TEG pH 8.0 RNase Ice Eppendorf centrifuge and Eppendorf tubes of 2 ml and 1.5 ml Pasteur pipettes Pipettes and disposable tips 1-5 ml, 100-1000 l. Vacuum desiccator
Method

1. Put the 7.5 M ammonium acetate solution on ice 2. Make 1 ml of fresh NaOH 0.2 M, SDS 1% solution by mixing the following solutions (in this order): 20 l 10 M NaOH 880 l milliQ water 100 l SDS 10% 3. Spin 1.5 ml o.n. culture of the bacteria 1 minute by 12000 rpm (Eppendorf centrifuge). 4. Discard the supernatant and fill again with 1.5 ml o.n. culture. Centrifuge, discard the supernatant and repeat this step again. Finally, you will have the pellet of 4.5 ml o.n. culture on the bottom of your Eppendorf tube. 5. Wash the pellet by resuspension in 0.5 ml STE. 6. Spin for 1 minute at 12,000 rpm. Discard the supernatant.

Laboratory Protocols
7. Resuspend the pellet in 200 l TEG 100 g/ml RNase. This step is crucial as total resuspension must be achieved in order to succeed in the isolation of the plasmid! 8. Incubate 5 minutes at room temperature. 9. Add 400 l of your freshly made NaOH 0.2M SDS 1 % solution and mix by hand. 10. Leave this for 5 to 10 minutes on crushed ice until you have a clear solution. 11. Add 300 l of ice cold 7.5 M ammonium acetate at pH 7.8 and mix well. 12. Leave for 10 minutes in ice water. 13. Spin for 5 minutes at 13,000 rpm in the Eppendorf centrifuge. 14. Transfer the clear supernatant over to a new Eppendorf tube and add 0.6 x isopropanol by volume. Mix by swirling and leave for 5 minutes at room temperature. 15. Centrifuge for 5 minutes at maximum rpm (Eppendorf centrifuge) and discard the supernatant with a pasteur pipette with a capillary ending. 16. Wash the pellet with 1 ml 70% ethanol and spin 2 minutes at maximum rpm in the Eppendorf centrifuge. Remove the ethanol; be careful not to remove the barely visible pellet. This will not be totally at the bottom but somewhat on the side (due to the centrifugal force) of the Eppendorf tube. 17. Remove the ethanol with a pasture pipette with a capillary ending. Let the rest of the alcohol damp away: use a vacuum desiccator for instance. The pellet should not become totally dry, because the last step is to redissolve it in 25 l TE-buffer. While adding the TE-buffer, be careful to let it flow over the pellet (with the DNA) in order to get the viscous, DNA-containing solution. Spin for 3 minutes at maximal rpm, then save on ice for instant use or put it in the freezer.