Latest revision as of 15:14, 5 August 2010

Making long term stocks | Transformation | [http://microscopy.berkeley.edu/Resources/instruction/buffers.html Buffer recipes] | Colony PCR

Transformation

Take competent cells out of -80 C freezer and thaw on ice (keep on ice at all times)
Cut yellow pipette tip(s) (as many as cell lines) with scalpel (after flaming with ethanol), cut onto a spatula
Transfer 50µl of cells to a separate 1.5 ml Eppendorff tube
Add 2µl of plasmid (if using pUC19 for testing competence)
Hold on ice for 30 mins
Heat shock at 42°C for 60 seconds using waterbath at back of Jim's lab ( must be switched on at start of expt)
On ice, add 250 ml SOC per tube (from bottle in 4°C fridge)
Put each Epp in top of 12ml falcon without lid, tape down.
Incubate at 37°C for 1 h with rotation (use rotating wheel incubator outside Jim's lab, reach around wheel and find switch to turn on/off)
Plate out 20µl (for TOP 10) or 100µl onto appropriate antibiotic plates with blue L-shaped spreader
Incubate overnight at 37°C

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+=Transformation=
+#Take competent cells out of -80 C freezer and thaw on ice (keep on ice at all times)
+#Cut yellow pipette tip(s) (as many as cell lines) with scalpel (after flaming with ethanol), cut onto a spatula
+#Transfer 50µl of cells to a separate 1.5 ml Eppendorff tube
+# Add 2µl of plasmid (if using pUC19 for testing competence)
+#Hold on ice for 30 mins
+# Heat shock at 42°C for 60 seconds using waterbath at back of Jim's lab (<strong> must be switched on at start of expt</strong>)
+# On ice, add 250 ml SOC per tube (from bottle in 4°C fridge)
+# Put each Epp in top of 12ml falcon without lid, tape down.
+# Incubate at 37°C for 1 h with rotation (use rotating wheel incubator outside Jim's lab, reach around wheel and find switch to turn on/off)
+# Plate out 20µl (for TOP 10) or 100µl onto appropriate antibiotic plates with blue L-shaped spreader
+#Incubate overnight at 37°C
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