Difference between revisions of "Genomic Insertion Protocol"
Wideloache (talk | contribs) |
Wideloache (talk | contribs) |
||
| Line 1: | Line 1: | ||
| + | ***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.*** | ||
| + | |||
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 |
