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Team:UNIPV-Pavia/Protocols/Digestion
From 2008.igem.org
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*To open vectors: | *To open vectors: | ||
| - | **1 µg of purified plasmid | + | **a volume containing 1 µg of purified plasmid |
**2 µl of buffer H | **2 µl of buffer H | ||
**1 µl of first enzyme | **1 µl of first enzyme | ||
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**incubate at 37°C for 3 hours | **incubate at 37°C for 3 hours | ||
*To excide fragments: | *To excide fragments: | ||
| - | **20 µl of purified plasmid when <7 µg | + | **20 µl of purified plasmid when <7 µg have been extracted. A volume containing 7 µg otherwise. |
**2.5 µl of buffer H | **2.5 µl of buffer H | ||
**1 µl of first enzyme | **1 µl of first enzyme | ||
Revision as of 12:30, 1 July 2008
 Home
|  The Team
|  The Project
|  Biological Safety
|  Parts Submitted to the Registry
|
|---|---|---|---|---|
 Dry Lab
|  Wet Lab
|  Modeling
|  Protocols
|  Activity Notebook
|
The protocols we used
- LB medium preparation
- Plasmid resuspension from IGEM paper spots
- Transformation
- Plasmid extraction
- BioBrick digestion with restriction enzymes
- DNA gel extraction
- 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
