Editing Fastbac Baculovirus Production

== Transformation<br>  ==

Transformation:<br> 

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

*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.<br>5. Heat-shock the cells for 45 seconds at 42 oC without shaking.<br>6. Immediately transfer the tubes to ice and chill for 2 minutes.<br>7. Add 900 ul of room temperature S.O.C. Medium.<br>8. For pFastBac transformations: Shake tubes at '''37 oC at 225 rpm for 4 hours'''.<br> For pUC19 transformation: Shake tube at 37 oC at 225 rpm for 1 hour.<br>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<br>LB agar plate containing 50 ug/ml '''kanamycin''', 7 ug/ml '''gentamicin''', 10 ug/ml '''tetracycline''', 100 ug/ml '''Bluo-gal''', and 40 ug/ml'''IPTG'''.<br>For the pUC19 transformation: Dilute the cells 1:100 with S. O.C. Medium.<br>9. Plate 100 ul of the dilution on an LB agar plate containing 100 ug/ml ampicillin.<br>10. Incubate plates for'''48''' hours at 37 oC. Pick '''white''' colonies for analysis.<br><br>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<br>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.<br><br> 

Clone Verfication<br> 

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<br> Bluo-gal, and 40 ug/ml IPTG. Incubate the plates overnight at 37 oC.<br>2. From a single colony confirmed to have a white phenotype on restreaked plates containing Bluo-gal and IPTG, inoculate a liquid culture containing<br> 50 ug/ml kanamycin, 7 ug/ml gentamicin, and 10 ug/ml tetracycline.<br>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<br> Plasmid Maxiprep Kit provided in the Appendix, page 44, for increased recombinant bacmid yield.<br>4. Analyze the recombinant bacmid DNA to verify successful transposition to the bacmid.<br><br><br> 

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[[Category:Protocols]]