https://eln.excellgen.com/index.php?action=history&feed=atom&title=Fastbac_Baculovirus_Production 2026-10-18T08:42:49Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=Fastbac_Baculovirus_Production&diff=6962&oldid=prev 2012-11-11T21:57:03Z

<p></p> <p><b>New page</b></p><div>== Transformation&lt;br&gt; ==<br /> <br /> Transformation:&lt;br&gt; <br /> <br /> 1. Thaw on ice one vial of DH10Bac competent E. coli cells for each transformation.&lt;br&gt;2. For each transformation, gently mix and transfer 100 ul of the DH10Bac cells into a pre-chilled, 15 ml round-bottom polypropylene tube.&lt;br&gt;3. Add the appropriate amount of plasmid DNA to the cells and mix gently. Do not pipet up and down to mix.&lt;br&gt; <br /> <br /> *Your recombinant pFastBac construct: 1 ng (5 ul) <br /> *pFastBac Gus control plasmid: 1 ng <br /> *pUC19 control: 50 pg (5 ul)<br /> <br /> 4. Incubate cells on ice for 30 minutes.&lt;br&gt;5. Heat-shock the cells for 45 seconds at 42 oC without shaking.&lt;br&gt;6. Immediately transfer the tubes to ice and chill for 2 minutes.&lt;br&gt;7. Add 900 ul of room temperature S.O.C. Medium.&lt;br&gt;8. For pFastBac transformations: Shake tubes at &#039;&#039;&#039;37 oC at 225 rpm for 4 hours&#039;&#039;&#039;.&lt;br&gt; For pUC19 transformation: Shake tube at 37 oC at 225 rpm for 1 hour.&lt;br&gt;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&lt;br&gt;LB agar plate containing 50 ug/ml &#039;&#039;&#039;kanamycin&#039;&#039;&#039;, 7 ug/ml &#039;&#039;&#039;gentamicin&#039;&#039;&#039;, 10 ug/ml &#039;&#039;&#039;tetracycline&#039;&#039;&#039;, 100 ug/ml &#039;&#039;&#039;Bluo-gal&#039;&#039;&#039;, and 40 ug/ml&#039;&#039;&#039;IPTG&#039;&#039;&#039;.&lt;br&gt;For the pUC19 transformation: Dilute the cells 1:100 with S. O.C. Medium.&lt;br&gt;9. Plate 100 ul of the dilution on an LB agar plate containing 100 ug/ml ampicillin.&lt;br&gt;10. Incubate plates for&#039;&#039;&#039;48&#039;&#039;&#039; hours at 37 oC. Pick &#039;&#039;&#039;white&#039;&#039;&#039; colonies for analysis.&lt;br&gt;&lt;br&gt;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&lt;br&gt;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.&lt;br&gt;&lt;br&gt; <br /> <br /> Clone Verfication&lt;br&gt; <br /> <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&lt;br&gt; Bluo-gal, and 40 ug/ml IPTG. Incubate the plates overnight at 37 oC.&lt;br&gt;2. From a single colony confirmed to have a white phenotype on restreaked plates containing Bluo-gal and IPTG, inoculate a liquid culture containing&lt;br&gt; 50 ug/ml kanamycin, 7 ug/ml gentamicin, and 10 ug/ml tetracycline.&lt;br&gt;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&lt;br&gt; Plasmid Maxiprep Kit provided in the Appendix, page 44, for increased recombinant bacmid yield.&lt;br&gt;4. Analyze the recombinant bacmid DNA to verify successful transposition to the bacmid.&lt;br&gt;&lt;br&gt;&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> [[Category:Protocols]]</div>

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