Valencia/Digestion
From 2007.igem.org
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===Standard Assembly=== | ===Standard Assembly=== | ||
| - | [[Image: | + | [[Image:VLupa.jpg ]]Standard Assembly is done in such way that the insert is intended to be larger than 500 pb. This means that if we are to ligate a promoter with a coding sequence, the promoter BioBrick will be the 'vector' and the coding sequence BioBrick will be the 'insert'. |
Usually we digest following the recommendations from the commercial information: | Usually we digest following the recommendations from the commercial information: | ||
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→ leave 2h at 37ºC, 180 rpm and then inactive 15 min at 65ºC | → leave 2h at 37ºC, 180 rpm and then inactive 15 min at 65ºC | ||
| - | [[Image: | + | [[Image:VLupa.jpg ]] |
| - | [[Image: | + | [[Image:VAdv.jpg ]] |
Latest revision as of 06:48, 26 September 2007
Information obtained from:
Roche Applied Science
We used four enzymes for the standard assembly:
- EcoRI
Protocol obtained from: EcoRI product information
Pdf link: Pdf
- XbaI
Protocol obtained from: XbaI product information
Pdf link: Pdf
- SpeI
Protocol obtained from: SpeI product information
Pdf link: Pdf
- PstI
Protocol obtained from: PstI product information
Pdf link: Pdf
Standard Assembly
Standard Assembly is done in such way that the insert is intended to be larger than 500 pb. This means that if we are to ligate a promoter with a coding sequence, the promoter BioBrick will be the 'vector' and the coding sequence BioBrick will be the 'insert'.
Usually we digest following the recommendations from the commercial information:
- BioBrick as vector (i.e., the part that, apart from the BioBrick, bears the origin of replication after the ligation)
| DNA | 1 ug |
| enzyme | 1 uL |
| enzyme | 1 uL |
| H buffer | 2 uL |
| ddwater | up to 20 uL |
→ leave 2h at 37ºC, 180 rpm and then inactive 15 min at 65ºC
- BioBrick as insert (i.e., part that only has the BioBrick)
| DNA | 1 ug |
| enzyme | 1uL |
| enzyme | 1 uL |
| H buffer | 2 uL |
| ddwater | up to 20 uL |
→ leave 2h at 37ºC, 180 rpm and then inactive 15 min at 65ºC
Changing the plasmid backbone
If we want to change the backbone (in order to change the antibiotic resistance) of a given plasmid, substituting the original BioBrick, for the new one, we will use EcoRI and PstI, instead of doing a Standard Assembly.
We follow this recipe:
- vector
| DNA | 1 ug |
| enzyme | 1 uL |
| enzyme | 1 uL |
| H buffer | 2 uL |
| ddwater | up to 20 uL |
→ leave 2h at 37ºC, 180 rpm and then inactive 15 min at 65ºC
- insert
| DNA | 1 ug | |
| enzyme | 1uL | |
| enzyme | 1 uL | |
| H buffer | 2 uL | |
| ddwater | up to 20 uL |
→ leave 2h at 37ºC, 180 rpm and then inactive 15 min at 65ºC
