Wisconsin/Protocol:Transformation

From 2007.igem.org

(Difference between revisions)
(Transforming Chemically Competent Cells)
(Transforming Chemically Competent Cells)
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===Transforming Chemically Competent Cells===
===Transforming Chemically Competent Cells===
#Thaw cells on ice
#Thaw cells on ice
-
#Pippet 100uL of cells into three 1.5mL tube. One for transformation and the other two for control.
+
#Pippet 100uL of cells into three ice-cold 1.5mL tube. One for transformation and the other two for control.
-
#*Normal: pippet 9uL of DNA into tube
+
#*Normal: pippet 9uL of ligation reaction into tube
#*Positive Control: pippet 2uL of pUC18 or pUC19 into tube
#*Positive Control: pippet 2uL of pUC18 or pUC19 into tube
#*Negative Control: nothing
#*Negative Control: nothing
#Sit on ice for 30 minutes
#Sit on ice for 30 minutes
-
#Heat shock in 42<sup>o</sup>C water bath for 30 seconds
+
#Heat shock in 42<sup>o</sup>C water bath for 90 seconds
#Incubate on ice for 2 minutes
#Incubate on ice for 2 minutes
-
#Transfer cells to new culture tubes (more O<sub>2</sub> for growth) and add 900uL of SOC to each culture tube
+
#Add 200uL of SOC to each culture tube
-
#Incubate on shaker at 37<sup>o</sup>C for 1 hour
+
#Incubate on shaker (nutator) at 37<sup>o</sup>C for 1 hour
#Grow 4 plates: 1 normal diluted, 1 normal concentrated, and 2 controls concentrated.
#Grow 4 plates: 1 normal diluted, 1 normal concentrated, and 2 controls concentrated.
-
#*Diluted: Spread 200uL onto plate
+
#*Diluted: Spread 50-100uL onto plate
-
#*Concentrated: Centrifuge tube for 5 minutes at 5000rpm first, then spread everything except for 200uL onto plates.
+
#*Concentrated: Centrifuge tube for 2-4 minutes at 5000rpm first, then spread everything except for 200uL onto plates.
#Incubate plates for 16 hours at 37<sup>o</sup>C. Plates should face down to prevent condensation on surface.
#Incubate plates for 16 hours at 37<sup>o</sup>C. Plates should face down to prevent condensation on surface.

Revision as of 16:23, 6 July 2007

Transforming Chemically Competent Cells

  1. Thaw cells on ice
  2. Pippet 100uL of cells into three ice-cold 1.5mL tube. One for transformation and the other two for control.
    • Normal: pippet 9uL of ligation reaction into tube
    • Positive Control: pippet 2uL of pUC18 or pUC19 into tube
    • Negative Control: nothing
  3. Sit on ice for 30 minutes
  4. Heat shock in 42oC water bath for 90 seconds
  5. Incubate on ice for 2 minutes
  6. Add 200uL of SOC to each culture tube
  7. Incubate on shaker (nutator) at 37oC for 1 hour
  8. Grow 4 plates: 1 normal diluted, 1 normal concentrated, and 2 controls concentrated.
    • Diluted: Spread 50-100uL onto plate
    • Concentrated: Centrifuge tube for 2-4 minutes at 5000rpm first, then spread everything except for 200uL onto plates.
  9. Incubate plates for 16 hours at 37oC. Plates should face down to prevent condensation on surface.