Bacterial Protocols

Contents

• Preparation of DH10BAC competent cells
• Preparation of transposition plates
• Transposition protocol
• Isolation of recombinant bacmid DNA
• Isolation of recombinant bacmid DNA (alternative protocol)
• Confirmation of transposition by PCR

Preparation of DH10BAC competent cells

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Stock solutions

LB Medium: Autoclave 1O g tryptone, 5 g yeast extract, and 5 g NaCl in 1 L.

Antibiotic solutions: To baculovirus resources

CaCl₂ solution: 60mM CaCl₂ 15%(v/v) glycerol, 10mM PIPES, pH adjusted to 7.0 with KOH. Filter sterilize.

Protocol

1. Inoculate a single colony of E.coli DH10BAC into 5mls of LB medium containing tetracycline at 10 µg/ml and kanamycin at 50 µg/ml. Grow overnight at 37°C with shaking (250 rpm).
2. Inoculate 4 mls of the culture into 400ml LB medium containing tetracycline at 10 µg/ml and kanamycin at 50 µg/ml in a 2 litre flask. Grow at 37°C with shaking (250rpm) to an OD₅₉₀ of 0.375. (This procedure requires that cells be growing rapidly ie. early or mid log phase).
3. Aliquot culture into 8 x 50ml pre-chilled, sterile polypropylene tubes and leave the tubes on ice 5 to 10mins. Centrifuge cells at 4000xg, 4°C for 15 mins.
4. Resuspend (gently) each pellet in 10ml ice cold CaCl₂ solution. Centrifuge 15 mins at 4000xg, 4°C.
5. Resuspend each pellet in 10mls cold CaCl₂ solution. Keep resuspended cells on ice for 30mins. Centrifuge 15 mins at 4000xg, 4°C.
6. Resuspend each pellet completely in 2mls of ice cold CaCl₂ solution. Aliquot 250 µl into prechilled, sterile polypropylene tubes. Freeze immediately at –70°C.

Preparation of transposition plates

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Stock solutions

Aqueous solutions of compounds should be sterilized by filtration through a 0.22 µm filter. Do not filter sterilize compounds dissolved in organic solvent. Store in small aliquots in light tight containers at -20°C. Magnesium ions are antagonists of tetracycline. Use media without magnesium salts for selection of bacteria resistant to tetracycline.

Plates

Autoclave L-Agar. Cool to 55°C and then add stock solutions.

Mix the agar solution before pouring plates under sterile conditions. Plates should be stored in the dark at 4°C, and are stable for up to four weeks.

Transposition protocol

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SOC Medium: 2% Bacto tryptone, 0.5% Bacto yeast extract, 10mM NaCL, 2.5mM KCl, 10mM MgCl₂, 10mM MgSO₄, 20mM glucose, filter sterilized.

1. Thaw the DH10BAC competent cells on ice.
2. Dispense 100 µl of the cells into 1.5 ml microfuge tubes.
3. Add 1 µg recombinant donor plasmid (in 5 µl) and gently mix the DNA with the cells.
4. Incubate the mixture on ice for 30 mins.
5. Heat shock the mixture by transferring to 42°C water bath for 45 secs.
6. Chill the mixture on ice for 2 mins.
7. Add 900 µl SOC medium to the mixture, and transfer culture to a universal.
8. Place the universal in a shaking incubator at 37°C with medium agitation (225 rpm) for 4-6 hours. The longer incubation period generates more white recombinant clones.
9. Serially dilute the cells, using SOC medium, to 10^-1, 10^-2 (i.e. 100 µl of transposition mix: 900 µl of SOC medium = 10^-1 dilution, use this to further dilute 10-fold to give 10^-2 dilution). Spin down 800 µl cells, discard 700 µl and resuspend in remaining 100 µl.
10. Place 100 µl of each dilution (resuspended, neat, 10^-1, 10^-2) on the plates and spread evenly over the surface.
11. Incubate for 48 hours at 37°C (colonies are very small and blue colonies may not be discernible prior to 24 hours).

Recombinant white and non recombinant blue colonies of E.coli DH10Bac. Competent DH10Bac cells were transformed with a recombinant pFastbac construct, plated out onto Bluogal containing media and incubated for 48 hours at 37°C. Cleavage of Bluogal by functional ß-galactosidase in non recombinants resulted in blue colonies. Transposition, however, caused insertional inactivation of the bacmid encoded alpha peptide gene, resulting in white colonies.

Isolation of recombinant bacmid DNA

White colonies contain the recombinant bacmid, and therefore, are selected for isolation of recombinant bacmid DNA. Before isolating DNA, candidate colonies are streaked to ensure they are truly white.

1. Pick 4 white colonies and streak to fresh plates to verify the phenotype. Incubate for 48 hours at 37°C.
2. From a single colony confirmed as having a white phenotype on plates containing Bluo-gal and IPTG, innoculate a 4 ml LB medium supplemented with 50 µg/ml kanamycin, 7 µg/ml gentamicin, and 10 µg/ml tetracycline. Grow at 37°C to stationary phase (up to 24 hours) shaking at 250 to 300 rpm.
3. Combine 0.9 ml recombinant bacmid culture with 0.1 ml sterile glycerol, and store at -80°C.
4. Transfer 1.5 ml of culture to a 1.5 ml microcentrifuge tube and centrifuge at 14,000xg for 1 min.
5. Remove the supernatant and resuspend each pellet in 0.3 ml of solution 1 (15 mM Tris HCL pH 8.0, 10 mM EDTA, 100 µg/ml RNase A). Add 0.3 ml of solution II (0.2 N NaOH, 1% SDS) and gently mix. Incubate at room temperature for 5 mins.
6. Slowly add 0.3 ml of 3 M potassium acetate pH 5.5, mixing gently during addition. A thick white precipitate of protein and E.coli genomic DNA will form. Place the sample on ice for 5 to 10 mins.
7. Centrifuge for 10 mins at 14,000xg. During the centrifugation, label another microcentrifuge tube and add 0.8 ml isopropanol to it. Gently transfer the supernatant to the tube containing isopropanol. Avoid any white precipitate material. Mix by gently inverting tube a few times and place on ice for 5 to 10 mins. At this stage, the sample can be store at -20°C overnight. Centrifuge the sample for 15 mins at 14,000 x g at room temperature.
8. Remove the supernatant and add 0.5 ml 70% ethanol to each tube. Centrifuge for 5 mins at 14,000 xg at room temperature.
9. Remove as much of the supernatant as possible.
10. Air dry the pellet briefly, 5 to 10 mins, at room temperature and dissolve the DNA in 40 µl sterile water. Store at -20°C.

Confirmation of transposition by PCR

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We routinely use the Expand High Fidelity PCR System cat. No. 1732650 from Roche Diagnostics.

*To prepare 2.5 mM dNTP mix, combine equal quantities of 10 mM dGTP, 10 mM dTTP, 10 mM dATP and 10 mM dCTP.

1. Set up PCR as above
2. Place tube in PCR machine with heated lid.
3. If the expected product is less than 5 kb, use the following cycling conditions: (93°C x 3 mins) x 1, (94°C x 45 secs, 55°C x 45 secs, 72°C x 5 mins) x 35. For larger products, add 1 minute extension time per kb of DNA.
4. Electrophorese 5 µl PCR on 1% agarose gel in electrophoresis buffer, and stain with ethidium bromide and visualize DNA under U.V. Light.

The expected results from the PCR are as follows:

Insertion of your gene into any of the pFastBac donor plasmids will result in an increase in the size of the PCR product. This increase in PCR product corresponds to the size of your gene of interest.