Revision as of 09:34, 17 June 2010

PCR purification protocol

Add 5 volumes of Buffer PBI to 1 volume of the PCR reaction and mix. If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.
Place a QIAquick column in a 2 ml collection tube.
To bind DNA, apply the sample to the QIAquick column and centrifuge for 30-60s. Discard flow-through and place the column back into the same tube.
To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30-60s. Discard flow-through and place the column back into the same tube.
Centrifuge the column in a 2 ml collection tube for 1 min.
Place each column in a clean 1.5 ml microcentrifuge tube.
To elute DNA, add 50 µl water ti the center of the QIAquick membrame and centrifuge the column for 1 min. For increased DNA concentration, add 30 ml elution buffer to the center of the QIAquick membrane, let the column st....

Thaw a 200µl aliquot of the desired strain of E. coli and add the transforming DNA (10 ng of vector DNA in 10 µl). Incubate for 45 mins at 42°C.
Heat-shock the cells for 120 secs, and place on ice again for 3-4 min.
Add 1ml of LB broth and incubate the cells at 37°C for 1-15 hr in a water bath with gentle shaking.
Plate out 100-200µl/plate on LB (agar at 1.5%), containing the appropriate selection markers.
Incubate plates overnight at 37°C.

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+{{Team:Newcastle/mainbanner}}
 =PCR purification protocol=
 # Add 5 volumes of Buffer PBI to 1 volume of the PCR reaction and mix. If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.
@@ Line 6: / Line 8: @@
 # Centrifuge the column in a 2 ml collection tube for 1 min.
 # Place each column in a clean 1.5 ml microcentrifuge tube.
-# To elute DNA, add 50 µl water ti the center of the QIAquick membrame and centrifuge the column for 1 min. For increased DNA concentration, add 30 ml elution buffer to the center of the QIAquick membrane, let the column st
+# To elute DNA, add 50 µl water ti the center of the QIAquick membrame and centrifuge the column for 1 min. For increased DNA concentration, add 30 ml elution buffer to the center of the QIAquick membrane, let the column st....
+=Transformation protocol=
+# Thaw a 200µl aliquot of the desired strain of ''E. coli'' and add the transforming DNA (10 ng of vector DNA in 10 µl). Incubate for 45 mins at 42°C.
+# Heat-shock the cells for 120 secs, and place on ice again for 3-4 min.
+# Add 1ml of LB broth and incubate the cells at 37°C for 1-15 hr in a water bath with gentle shaking.
+# Plate out 100-200µl/plate on LB (agar at 1.5%), containing the appropriate selection markers.
+# Incubate plates overnight at 37°C.
+{{Team:Newcastle/footer}}