Difference between revisions of "Berk2010-Tim"

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(Tim Hsiau 20:13, 4 June 2010 (EDT))
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PDD's are not stable, need to make competent cells of BAC plus payload and do fresh transforms of PDD's everytime you assay
 
PDD's are not stable, need to make competent cells of BAC plus payload and do fresh transforms of PDD's everytime you assay
  
We have to work with MG1655  
+
We have to work with MG1655 for assays, other strains okay for cloning
  
 
Other notes:
 
Other notes:

Revision as of 17:30, 4 June 2010

Tim Hsiau 20:13, 4 June 2010 (EDT)

Talked to Jin about payload delivery device

Jin Notes.JPG

first column is PDD: payload delivery device

second column is the payload plus some stuff that may inhibit vacuole merging (secretion system)

third column is the BAC that contains other secretion system components

PDD's are not stable, need to make competent cells of BAC plus payload and do fresh transforms of PDD's everytime you assay

We have to work with MG1655 for assays, other strains okay for cloning

Other notes:

Mg needs to be added for all steps of cloning the PDD's
1M MgSO4
10µL into rescue step (after heatshock)
400µL spread on plate (if +Spec, add 20µL and spread)
30µL into ea mL TB media (use TB for growing up for assay)