Latest revision as of 11:44, 22 May 2008

Transformation

Shock Cells, monitor discharge time
Add SOC to cuvettes (~400)
Transfer to culture tubes, shake at 37 for 1 hour (depending on antibiotic marker)
Plate ~200uL of SOC/Cells onto agar plate

¹To make SOC, add 20 μL 500 mM filter-sterilized glucose to 500 uL SOB (20 mM final glucose concentration)

@@ Line 1: / Line 1: @@
 ='''Transformation'''=
-''Time: 2.25 hr''
 ===<font color="blue">Media/Reagents</font color>===
@@ Line 8: / Line 7: @@
 ===<font color="red">Pre</font>===
-*Chill ligated DNA on ice
+*Add plasmid DNA to alloquats from electrocompetent cell prep
-*Thaw competent cells ((found in eppendorfs in box on second-shelf down of -80ºC freezer) on ice for ~5 min.
+*Transfer DNA and Cells into electroporator cuvettes
-===<font color="green">Transformation</font color>===
+===<font color="green">Electroporation</font color>===
-#Add 3 μL ligation mixture to cells in bottom of tube
+#Shock Cells, monitor discharge time
-#Incubate on ice for 30 min
+#Add SOC to cuvettes (~400)
-#Heat shock in 42ºC water bath for 1 min (warm SOC media at same time).
+#Transfer to culture tubes, shake at 37 for 1 hour (depending on antibiotic marker)
-#Incubate on ice for 2 min
+#Plate ~200uL of SOC/Cells onto agar plate
-#Add 500 μL warm SOC media
-#Shake in 37ºC room for 1 hr
-#Spread 280 μL on LB/antibiotic plates using rotator and glass spreading wand.
-#Incubate plates in 37ºC room overnight.
+<sup>1</sup>To make SOC, add 20 μL 500 mM filter-sterilized glucose to 500 uL SOB (20 mM final glucose concentration)
-<sup>1</sup>To make SOC, add 200 μL 500 mM filter-sterilized glucose to 5 mL SOB (20 mM final glucose concentration)
-[http://openwetware.org/wiki/Penn_State_University_2006:Cloning Cloning]
-[http://openwetware.org/wiki/IGEM:PennState Main]