Team:Purdue/E. coli transformation
From 2010.igem.org
(Difference between revisions)
(New page: Transformation of E. Coli - Obtained materials – buckets of ice, 5 ml SOC, X21 Gold Competent Cells, Kan Plates, hot water bath, shaker in 37 degree room - Took XL-21 gold from -80, adde...) |
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- | + | ==Materials== | |
- | + | *buckets of ice | |
- | + | *5 ml SOC | |
- | + | *X21 Gold Competent Cells | |
- | + | *Kan Plates | |
- | + | *hot water bath | |
- | + | *shaker in 37 degree room | |
- | + | ==Procedure== | |
- | + | #Took XL-21 gold from -80, added 12μl to transformation tube and moved immediately to ice bucket | |
- | + | #Added 1.5 μl luciferase plasmid DNA (with kanamycin resistance) to eppendorf tube | |
- | + | #Prepared a control without the plasmid | |
- | + | #Stored on ice for 30 minutes | |
+ | #Heat shock in 42 degree water bath for 28 sec | ||
+ | #Moved to ice bucket for 2 minutes | ||
+ | #Added 200μl SOC carefully to the control and the transformant | ||
+ | #Moved the tubes to 37 degree shaker, set at 100 rpm, tilted at an angle for better mixing – for one hour | ||
+ | #Obtained three kan plates and plated 50 μl control, 50 μl transform, and 100 μl of transform – streaked with glass beads | ||
+ | #Stored plates in 37 degree room for 11 hours |
Latest revision as of 14:28, 13 July 2010
Materials
- buckets of ice
- 5 ml SOC
- X21 Gold Competent Cells
- Kan Plates
- hot water bath
- shaker in 37 degree room
Procedure
- Took XL-21 gold from -80, added 12μl to transformation tube and moved immediately to ice bucket
- Added 1.5 μl luciferase plasmid DNA (with kanamycin resistance) to eppendorf tube
- Prepared a control without the plasmid
- Stored on ice for 30 minutes
- Heat shock in 42 degree water bath for 28 sec
- Moved to ice bucket for 2 minutes
- Added 200μl SOC carefully to the control and the transformant
- Moved the tubes to 37 degree shaker, set at 100 rpm, tilted at an angle for better mixing – for one hour
- Obtained three kan plates and plated 50 μl control, 50 μl transform, and 100 μl of transform – streaked with glass beads
- Stored plates in 37 degree room for 11 hours