Latest revision as of 12:52, 1 July 2008

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The protocols we used

LB medium preparation
Plasmid resuspension from IGEM paper spots
Transformation
Plasmid extraction
BioBrick digestion with restriction enzymes
DNA gel extraction
DNA precipitation with sodium acetate
Antarctic Phosphatase
Ligation
PCR

BioBrick digestion with restriction enzymes

(estimated time: 3 hours)

Materials needed:

Roche restriction enzymes thawed on ice
Roche buffer H
Pre-warmed at 37°C bath
Cut and gel-extracted vector
ddH2O

To open vectors:
- a volume containing 1 µg of purified plasmid
- 2 µl of buffer H
- 1 µl of first enzyme
- 1 µl of second enzyme
- 20 µl final volume
- incubate at 37°C for 3 hours
To excide fragments:
- 20 µl of purified plasmid when <7 µg have been extracted. A volume containing 7 µg otherwise.
- 2.5 µl of buffer H
- 1 µl of first enzyme
- 1 µl of second enzyme
- 25 µl final volume
- incubate at 37°C for 2 hours and 30 minutes

@@ 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 28: / Line 29: @@
 <h1>BioBrick digestion with restriction enzymes</h1>
-''(estimated time: 2 hours and 15 min)''
+''(estimated time: 3 hours)''
 <br>
 <br>
 '''Materials needed:'''
-*'''Roche'''
+*'''Roche restriction enzymes thawed on ice'''
-*'''Pre-warmed at bath'''
+*'''Roche buffer H'''
+*'''Pre-warmed at 37°C bath'''
 *'''Cut and gel-extracted vector'''
+*'''ddH2O'''
 <br>
-*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).
+*To open vectors:
-*Add 1 µl of Antarctic Phosphatase (up to 5 µg of cut vector).
+**a volume containing 1 µg of purified plasmid
-*Incubate at 37°C for 1 hour (Antarctic Phosphatase works).
+**2 µl of buffer H
-*Incubate at 65°C for 15 min (Antarctic Phosphatase inactivation).
+**1 µl of first enzyme
+**1 µl of second enzyme
+**20 µl final volume
+**incubate at 37°C for 3 hours
+*To excide fragments:
+**20 µl of purified plasmid when <7 µg have been extracted. A volume containing 7 µg otherwise.
+**2.5 µl of buffer H
+**1 µl of first enzyme
+**1 µl of second enzyme
+**25 µl final volume
+**incubate at 37°C for 2 hours and 30 minutes
 <br>