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Team:UNIPV-Pavia/Protocols/Digestion
From 2008.igem.org
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*[[Team:UNIPV-Pavia/Protocols/Digestion|BioBrick digestion with restriction enzymes]] | *[[Team:UNIPV-Pavia/Protocols/Digestion|BioBrick digestion with restriction enzymes]] | ||
*[[Team:UNIPV-Pavia/Protocols/GelExtraction|DNA gel extraction]] | *[[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/AntarcticPhosphatase|Antarctic Phosphatase]] | ||
*[[Team:UNIPV-Pavia/Protocols/Ligation|Ligation]] | *[[Team:UNIPV-Pavia/Protocols/Ligation|Ligation]] | ||
| Line 40: | Line 41: | ||
<br> | <br> | ||
*To open vectors: | *To open vectors: | ||
| - | **20 final volume | + | **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 | ||
<br> | <br> | ||
Latest revision as of 12:52, 1 July 2008
 Home
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|
|---|---|---|---|---|
 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
- 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
