Latest revision as of 12:53, 1 July 2008

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The protocols we used

(estimated time: 1 hour and 30 min)

Materials needed:

Add the proper amount of 10X buffer to a final concentration of 1X (e.g. 2 µl of 10X buffer in a final volume of 20 µl).
Add 1 µl of Antarctic Phosphatase (up to 5 µg of cut vector).
Incubate at 37°C for 1 hour (Antarctic Phosphatase works).
Incubate at 65°C for 15 min (Antarctic Phosphatase inactivation).

@@ Line 22: / Line 22: @@
 *[[Team:UNIPV-Pavia/Protocols/Digestion|BioBrick digestion with restriction enzymes]]
 *[[Team:UNIPV-Pavia/Protocols/GelExtraction|DNA gel extraction]]
+*[[Team:UNIPV-Pavia/Protocols/Precipitation|DNA precipitation with sodium acetate]]
 *[[Team:UNIPV-Pavia/Protocols/AntarcticPhosphatase|Antarctic Phosphatase]]
 *[[Team:UNIPV-Pavia/Protocols/Ligation|Ligation]]
@@ Line 37: / Line 38: @@
 *'''Cut and gel-extracted vector'''
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-*Add the proper amount of 10X buffer to a final concentration of 1X (e.g. 2 )
+*Add the proper amount of 10X buffer to a final concentration of 1X (e.g. 2 µl of 10X buffer in a final volume of 20 µl).
-*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.
+*Add 1 µl of Antarctic Phosphatase (up to 5 µg of cut vector).
-*Put the cut paper into the 0.5 ml tube.
+*Incubate at 37°C for 1 hour (Antarctic Phosphatase works).
-*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 much more time to dry than scalpel/tweezers.
+*Incubate at 65°C for 15 min (Antarctic Phosphatase inactivation).
-*Incubate at 42°C for 20 min.
-*Vortex and spin down.
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