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Lab Protocols

Bacmid Transformation and Purification by Arthur Laganowsky

Adapted from M. Faller/Guo Lab

 

pBAC Plasmid Transformation

  1. Thaw one tube (or 100 uL) of Max Efficiency Dh10Bac chemically competent cells on ice. Label four 1.5 mL centrifuge tubes and place on ice, as well. Usually about 8-10 minutes.

  2. Keep everything on ice. Add 1uL of pBAC-construct* to each 1.5mL centrifuge tubes.

  3. Gently add 22 uL of competent cells to each tube. Let sit on ice for 30 minutes.

  4. Heat shock for 45 seconds at 42 C.

  5. Immediately place on ice for 2 minutes.

  6. Add 500 uL of pre-warmed (37 C) SOC media.

  7. Incubate in 37 C shaker for four hours.

  8. After four hours of incubation, prepare eight tubes for two dilutions (1:1000, 1:100):

      a. For 1:1000, add 999 uL SOC and 1 uL cells

b. For 1:100, add 990 uL SOC and 10 uL cells

  1. Spread 100 uL of diluted cells onto Bacmid plates.

  2. Incubate at 37 for two days. White colonies contain insert.^

* pBAC-construct should be sequenced prior to transformation.

^ Check by colony pcr or after bacmid preparation by pcr if bacmid contains target gene.

 

Bacmid Purification

  1. Inoculate a single, isolated bacterial colony in 2 mL of LB medium containing 50ug/mL kanamycin, 7ug/ml gentamicin, and 10 ug/mL tetracycline. Usually two colonies per clone.

  2. Grow overnight culture in 37 C shaker.

  3. Transfer 1.5 mL of bacterial culture to a 1.5 mL centrifuge tube and centrifuge at 14,000 x g for 1 minute to pellet cells.

  4. Remove the supernatent and resuspend cell pellet in 0.3 mL (0.6 mL) Solution I. Gently vortex or pipet up and down to resuspend.

  5. Add 0.3 mL of Solution II and gently mix. Incubate at room temperature for 5 minutes. Note: The appearance of the suspension should change from turbid to almost translucent.

  6. Slowly add 0.3 mL of solution 3, mixing gently during addition. A thick white percipitate of protein and E. coli genomic DNA will form. Place the sample on ice for 5-10 minutes.

  7. Centrifuge for 10 minutes at 14,000 x g.

  8. Gently transfer the supernatent to a microcentrifuge tube containing 0.8 mL of isopropanol. Do not transfer any white percipitate. Invert the tube a few times to mix and place on ice for 5-10 minutes. Proceed directly to Step 9 or store sample at -20 C overnight.

  9. Centrifuge the sample for 15 minutes at 14,000 x g at room temperature.

  10. Carefully remove the supernatent, taking care not to disturb the pellet. Add 0.5mL of cold 70% ethanol. Invert the tube several times to wash the pellet.

  11. Centrifuge for 5 minutes at 14,000 x g at room temperature. Repeat Steps 10 and 11, if desired.

  12. Decant supernatent, and air dry pellet.

  13. Resuspent with 25uL sterile water.

  14. Do not freeze the bacmid, as it may lead to the shearing of the DNA. It can be stored at 4 C for short period or precipitated in ethanol at -20 C.

Solution 1 ~ 50 mM Tris-HCl pH 8.0, 10mM EDTA, 100 ug/mL RNase A

Solution 2 ~ 200 mM NaOH, 1% SDS

Solution 3 ~ 3.3 M Potassium Acetate pH 5.5

 

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