Fastbac Baculovirus Production

Transformation

Transformation:

1. Thaw on ice one vial of DH10Bac competent E. coli cells for each transformation.
2. For each transformation, gently mix and transfer 100 ul of the DH10Bac cells into a pre-chilled, 15 ml round-bottom polypropylene tube.
3. Add the appropriate amount of plasmid DNA to the cells and mix gently. Do not pipet up and down to mix.

• Your recombinant pFastBac construct: 1 ng (5 ul)
• pFastBac Gus control plasmid: 1 ng
• pUC19 control: 50 pg (5 ul)

4. Incubate cells on ice for 30 minutes.
5. Heat-shock the cells for 45 seconds at 42 oC without shaking.
6. Immediately transfer the tubes to ice and chill for 2 minutes.
7. Add 900 ul of room temperature S.O.C. Medium.
8. For pFastBac transformations: Shake tubes at 37 oC at 225 rpm for 4 hours.
For pUC19 transformation: Shake tube at 37 oC at 225 rpm for 1 hour.
For each pFastBac transformation: Prepare 10-fold serial dilutions of the cells (10–1, 10–2, 10–3) with S.O.C. Medium. Plate 100 ul of each dilution on an
LB agar plate containing 50 ug/ml kanamycin, 7 ug/ml gentamicin, 10 ug/ml tetracycline, 100 ug/ml Bluo-gal, and 40 ug/mlIPTG.
For the pUC19 transformation: Dilute the cells 1:100 with S. O.C. Medium.
9. Plate 100 ul of the dilution on an LB agar plate containing 100 ug/ml ampicillin.
10. Incubate plates for48 hours at 37 oC. Pick white colonies for analysis.

Note: Do not picking colonies earlier than 48 hours as it may be difficult to distinguish between white and blue colonies. True white colonies tend
to be large; therefore, to avoid selecting false positives, choose the largest, most isolated white colonies. Avoid picking colonies that appear gray or are darker in the center as they can contain a mixture of cells with empty bacmid and recombinant bacmid.

Clone Verfication

1. Pick 10 white colonies and restreak them on fresh LB agar plates containing 50 ug/ml kanamycin, 7 ug/ml gentamicin, 10 ug/ml tetracycline, 100 ug/ml
Bluo-gal, and 40 ug/ml IPTG. Incubate the plates overnight at 37 oC.
2. From a single colony confirmed to have a white phenotype on restreaked plates containing Bluo-gal and IPTG, inoculate a liquid culture containing
50 ug/ml kanamycin, 7 ug/ml gentamicin, and 10 ug/ml tetracycline.
3. Isolate recombinant bacmid DNA using the procedure provided on the next page for analysis. You may also use the procedure for the PureLinkTM HiPure
Plasmid Maxiprep Kit provided in the Appendix, page 44, for increased recombinant bacmid yield.
4. Analyze the recombinant bacmid DNA to verify successful transposition to the bacmid.