Revision as of 23:25, 9 June 2008

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Transformation

(estimated time: 3 hours and 30 min + 12-16 hours overnight incubation)

Materials needed:

LB agar plates with proper antibiotic added incubated at 37°C
Thawed Invitrogen TOP10 cells (every tube contains approximately 60 µl of competent cells)
Resuspended DNA
SOC medium

Put 4-6 µl DNA resuspension into TOP10 tube.
Incubate on ice for 30-45 min.
Heat shock: 42°C for 1 min.
Put transformed TOP10 tube on ice and then add 250 µl SOC medium.
Incubate 2 hours at 37°C, 220 rpm.
Centrifuge 10 min, 1200 rpm.
Remove 150 ul of bacteria-free supernatant.
Plate the remaining part of solution (resuspending the bacteria) on a proper agar plate.
Incubate overnight at 37°C.

@@ Line 16: / Line 16: @@
 <h1>Transformation</h1>
-''(estimated time: 3 hours and 30 min + 12-14 hours overnight incubation)''
+''(estimated time: 3 hours and 30 min + 12-16 hours overnight incubation)''
 <br>
 <br>
 '''Materials needed:'''
 *'''LB agar plates with proper antibiotic added incubated at 37°C'''
-*'''Thawed Invitrogen TOP10 cells (every tube contains approximately 60 )'''
+*'''Thawed Invitrogen TOP10 cells (every tube contains approximately 60 µl of competent cells)'''
 *'''Resuspended DNA'''
 *'''SOC medium'''
 <br>
-*
+*Put 4-6 µl DNA resuspension into TOP10 tube.
-*Cut paper spots using scalpel and tweezers (or punch tool, following provided instructions); if you use scalpel and tweezers, try to cut pieces of about the same dimension of the punch tool.
+*Incubate on ice for 30-45 min.
-*Clean scalpel and tweezers (or punch tool) with water and ethanol every time you cut a spot; be careful to dry your tools correctly, especially if you use punch tool, which needs more time to dry than scalpel/tweezers.
+*Heat shock: 42°C for 1 min.
-*Put the cut paper into the 0.5 ml tube.
+*Put transformed TOP10 tube on ice and then add 250 µl SOC medium.
-*Incubate at 42°C for 20 min.
+*Incubate 2 hours at 37°C, 220 rpm.
-*Vortex and spin down.
+*Centrifuge 10 min, 1200 rpm.
+*Remove 150 ul of bacteria-free supernatant.
+*Plate the remaining part of solution (resuspending the bacteria) on a proper agar plate.
+*Incubate overnight at 37°C.
 <br>