BAC/Fosmid [Alkaline Lysis] Plasmid Prep

1. Inoculate a fresh single colony into 50-500ml 2xYT in a 500ml Erlenmeyer flask. More cells are required depending on the Plasmid copy number. If in doubt, use at least 250 ml of overnight. Normal LB can be substituted for YT. Grow for 20+ hours with agitation ~300 r.p.m.

2. Transfer into 50ml centrifuge tubes. Pellet at 4500 r.p.m. for 30 minutes at 4C.

3. Carefully pour off supernatant. Invert for 5 minutes on paper towel to dry. Place tubes on ice and add 5ml of cooled (4C) GET buffer. Add 60uL of 20mg/ml RNAseA. Resuspend by stirring with a sealed end Pasteur pipette or other sterile implement. This is to loosen the pellet, gently scrape the cells from the walls of the centrifuge tubes. Follow by repeatedly pipetting up and down to ensure complete resuspention.

4. Add 5ml lysis solution (0.2M NaOH + 1% SDS) to the samples. Invert very gently 2 times. Add 5ml 3M KOAc pH 5.5 Invert 2 times. Place on ice. Repeat for all samples. Incubate cells on ice for 5 minutes. Invert 5 times. Incubate for a further 10 minutes. Invert 5 times.

5. Spin at 15 000 r.p.m. for 30 minutes at 4C.

6. If pellet solid carefully transfer the supernatant by pipetting (with Pseudomonas Putida, do not do more then 200uL at a time as precipitant very loose even after extensive centrifuging). If not, spin at 16 000 for 10 more minutes. Make sure that supernatant is free of any white colored precipitant after transfer. Pool all samples.

7. Add 0.67 volumes of isopropanol, this depends on the volume of supernatant retrieved from the previous step. Incubate for 15 minutes at room temperature.

8. Spin at 15 000 for 30 minutes at 10-15C.

9. Carefully remove supernatant. The pellet may be loose. Fill tube with 70% ethanol, pouring the ethanol down the opposite side of the tube as the pellet. Pour off ethanol, if the looks too loose then spin at 15 000 for an additional 15 minutes.

10. Once the ethanol has been removed, cover the top with parafilm, poke holes in it and dry samples
in the speed vac for 15 minutes. Ensure that the pellet is dry before the next step.

11. While the tube is on ice add 500uL of TE buffer 10:1 pH 8.0. Allow the sample to Resuspend for 30 minutes while on ice. Mix by swirling the tube, careful vortexing or repeated pipetting. Transfer to a 1.5 mL eppendorf tube.

12. Add 700 uL phenol-chloroform, vortex and ice for 5 minutes then add 700 uL chloroform. Spin for 10 minutes and extract top layer into a fresh 1.5 mL tube. Add 700 uL chloroform, vortex and spin for 10 minutes. Extract top layer into a fresh 1.5 ml tube.

13. Add 700uL isopropanol, vortex. Spin for 15 minutes to pellet DNA.

14. Extract the IPA with a Pasteur pipette and bulb. Be careful of the pellet, it will be loose. Do not pour off the IPA.

15. Add 1mL 70% ethanol opposite the side of the pellet. Spin for 5 minutes. Extract the ethanol with a Pasteur pipette and bulb. Dry samples in speed vac.

16. Resuspend sample in 40uL of TE 10:1 pH 8.0. Visualize on a 0.4% agarose gel at 20 V overnight.

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Cesium Chloride Plasmid Prep Protocol

1. Spin 250ml of overnight at 7 000 r.p.m. for 10 minutes. Resuspend in 5ml glucose buffer.

2. Add 1mL glucose buffer with 20mg/ml lysozyme + 2mg/ml pronase. Incubate for 5 minutes at room temperature.

3. Lyse with 12mL lysing solution (2% SDS, 0.4M NaOH). Gently swirl and place on ice 5 minutes.

4. Add 5ml Sodium Acetate, incubate for 15 minutes. Spin out insolubles at 10 000 r.p.m. Pour supernatant into 40ml tubes. Extract with phenol/chloroform/isoamylalcohol (25:24:1) Spin at 7 000 for 10 minutes.

5. Add 1.1g Cesium Chloride and 1mL 65% CsCl in TE. Spin at 90 000 for 4 hours at 18C.

6. Remove supernatant, dialyze for 6 hours.

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Hydrocarbon Layer Deposition

1. Make stock Eicosane solution 0.01g/ml in ether.

2. Prepare M9 media plates.

3. Using narrow bore syringe spray plates 250uL at a time with stock Eicosane solution. Repeat 4 times for a total volume of 1000uL. Spray in fume hood at a high of 20-30cm above plate, depending on temperature and flow rate of fume hood fan.

4. Or place plate on plate spinner in fume hood, use glass pipette to pipette 1000uL of stock solution onto rapidly spinning plate. Very thin layer should form. Spin for at least 10 seconds to ensure complete evaporation of ether solvent.

5. Syringe method produces more consistent zones of clearing due to thin layer but pour method produced very even layers, which are easier to visualize.

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Qiagen Column DNA Purification Protocol

1. Centrifuge cells at 13 000 r.p.m. for 10 minutes.

2. Remove supernatant and Resuspend pelleted bacterial cells in 250 uL P1 Buffer and transfer into microcentrifuge tube.

3. Add 250 uL P2 Buffer and gently invert the tube 4-6 times to mix.

4. Add 350 uL N3 Buffer and invert tube immediately but gently 4-6 times.

5. Centrifuge at 13 000 r.p.m. for 10 minutes.

6. Apply supernatant (contains the DNA) onto QIAprep spin column by pipetting.

7. Centrifuge for 60 seconds, discard flow-through.

8. Wash column with 0.5ml PB Buffer and centrifuge for 60 seconds. Discard flow-through.

9. Wash column by adding 0.75ml PE Buffer and centrifuge for 60 seconds. Discard flow-though and centrifuge for additional 60 seconds to remove residual buffer.

10. Place column into clean 1.5ml microcentrifuge tube. To elute DNA add 50uL EB Buffer (Elution Buffer) 10mM Tris-Cl pH 8.5. Incubate for 60 seconds at room temerature. Centrifuge for 60 seconds at 13 000.

11. Microcentrifuge contents contain purified DNA.

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TOPO Cloning Kit Protocol

1. Produce PCR products using Taq Polymerase. End with 10-minute extension step to ensure reaction runs to completion.

2. Set up TOPO cloning reaction:
- 0.5-4.0uL PCR product
- 1uL Salt solution
- 1uL TOPO Vector
- Sterile H20 to final total volume of 5uL

3. Mix gently and incubate for 30 seconds to 30 minutes at room temperature. Longer incubation times improved success.

4. Place tubes on ice. Proceed with One Shot Chemical Transformation.

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One Shot E.Coli Chemical Transformation

1. 2uL TOPO reaction product into one vial of one shot chemically competent E. Coli.

2. Incubate on ice for 30 minutes.

3. Heat shock at 42C for 30 seconds.

4. Add 250 uL S.O.C. medium

5. Shake at 37C for 1 hour
6. Plate.

7. Incubate overnight at 37C on kanamycin antibiotic plates.

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PCR

- 5uL PCR Buffer
- 2uL MgCl2
- 1uL dNTP’s
- 0.5uL AlkB-up
- 0.5uL AlkB-down
- 0.5uL Taq
- 40uL DH2O
- 0.5uL Template cells

Total volume = 50uL
36 Cycles.

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Gradient PCR

In order to produce a wide range of point mutants with varying degrees of mutation, gradient PCR was used. The volume of 50mM MgCl2 was varied from 0uL to 12uL per 50uL PCR reaction in 0.5mM increments. The optimal MgCl2 volume is somewhere between 5uL and 10uL. The plateau is quite wide with similar product yields within the 5-10uL range.

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