Difference between revisions of "Genomic Insertion Protocol"

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***This protocol needs to be modified an updated based on the results obtained in my first attempt at genomic integration. Please see my [http://gcat.davidson.edu/GcatWiki/index.php/Will_DeLoache_Notebook lab notebook results] until then.***
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Note that you'll need a pir strain for replication of pG80 plasmids.  You can drop your biobrick into the Eco/pst sites of pG80ko, transform the pir116 cells.  Grow up, miniprep, and map a single colony.  Make competent cells of pInt80-649 in your target strain (plate them on Amp).  It is temperature sensitive, so do all growth manipulations at 30 degrees.  Transform in your pG80 derivative, plate on 15ug/mL gentamicin plates at 37 degrees.  Grow a single colony to saturation at 37 in LB+15ug/mL gentamicin, then restreak on a gentamicin plate at 43 degrees.  You can use the oligos below to PCR amplify the phi80 locus for confirmation of integration and sequencing.
 
Note that you'll need a pir strain for replication of pG80 plasmids.  You can drop your biobrick into the Eco/pst sites of pG80ko, transform the pir116 cells.  Grow up, miniprep, and map a single colony.  Make competent cells of pInt80-649 in your target strain (plate them on Amp).  It is temperature sensitive, so do all growth manipulations at 30 degrees.  Transform in your pG80 derivative, plate on 15ug/mL gentamicin plates at 37 degrees.  Grow a single colony to saturation at 37 in LB+15ug/mL gentamicin, then restreak on a gentamicin plate at 43 degrees.  You can use the oligos below to PCR amplify the phi80 locus for confirmation of integration and sequencing.
  

Revision as of 02:32, 16 March 2009

      • This protocol needs to be modified an updated based on the results obtained in my first attempt at genomic integration. Please see my lab notebook results until then.***

Note that you'll need a pir strain for replication of pG80 plasmids. You can drop your biobrick into the Eco/pst sites of pG80ko, transform the pir116 cells. Grow up, miniprep, and map a single colony. Make competent cells of pInt80-649 in your target strain (plate them on Amp). It is temperature sensitive, so do all growth manipulations at 30 degrees. Transform in your pG80 derivative, plate on 15ug/mL gentamicin plates at 37 degrees. Grow a single colony to saturation at 37 in LB+15ug/mL gentamicin, then restreak on a gentamicin plate at 43 degrees. You can use the oligos below to PCR amplify the phi80 locus for confirmation of integration and sequencing.

attPhi80-1: CTGCTTGTGGTGGTGAAT

attPhi80-2: ACTTAACGGCTGACATGG

attPhi80-3: ACGAGTATCGAGATGGCA

attPhi80-4: TAAGGCAAGACGATCAGG

sequence Temp (°C) No integrant with 1 and 4 Single integrant with 1 and 2, 3 and 4 Multiple integrant with 1 and 2, 3 and 2, 3 and 4
63 546 409, 732 409, 595, 732