Difference: ProtocolsAcinetobacterTransformation (8 vs. 9)

Revision 92019-05-29 - GabrielSuarez

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Transforming Acinetobacter baylyi ADP1

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  1. Pick colonies that grow on selective media and use PCR to assay for a successful transformation. Growth on non-selective but not selective plating suggests a failed transformation.

Notes/Considerations in ADP1 transformations

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  • Growth and transformation occur with equal transformation frequency on appropriate minimal medias as they do in LB (examples: M9+1% glucose, S2 lactic acid media, and succinate minimal media).
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  • Growth and transformation occur with equal transformation frequency on appropriate minimal medias as they do in LB (examples: M9+1% glucose, S2 lactic acid media, and succinate minimal media - the current standard ProtocolsRecipesABMS).
 
  • If using 100ng of PCR product with at least 1000bp flanking homology on either side of the transformed marker or ADP1 gDNA, you can expect transformation frequencies of around 10E-4.
  • Transformation frequency can be increased by increasing the concentration of transforming DNA, with a maximum transformation frequency (approx. 10E-3 to 10E-2) at 2ug/mL of ADP1 genomic DNA.
  • Transformation frequency is sensitive to the amount of homology a construct has to the ADP1 genome for PCR products. 500bp on each flank should be used as a minimum for moderate efficiency (10E-5 transformation frequency at 100ng per 1mL transformation) and a minimum of 250bp on each flank should be used in general, as transformations with less homology are unreliable.
 
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