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Team:Warsaw/Calendar-Main/18 July 2009
From 2009.igem.org
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Insertion of the pho gene into the pKSII+ plasmid
Kama
- Plasmid digest mix was prepared as follows:
2μl Tango buffer (Fermentas) 1μl pKSII plasmid 0,5μl XbaI enzyme (Fermentas) 0,5μl SmaI enzyme (Fermentas)
The solution was topped up with H2O to the final volume of 20 μl. - Pho gene digest mix was prepared as follows:
2μl Tango buffer (Fermentas) 9μl purified gene 0,5μl XbaI enzyme (Fermentas)
The solution was topped up with H2O to the final volume of 20 μl. - The digest was kept for 3h at 37°C, and then the enzymes were inactivated for 20min. at 80°C.
The ligation mix was prepared as follows:
20μl plasmid 20μl gene 5μl ligation buffer with PEG 1 μl T4 DNA ligase (Fermentas)
The solution was topped up with H2O to the final volume of 50 μl. The ligation was carried out in 18°C overnight (~15h)
Cloning of p53 coding sequence
Marcin
Task:
- Isolation of plasmid containing p53 CDS and digest them using PvuII restriction enzyme
Methods:
- Plasmids weren isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here.
- Digest of pKS/p53 plasmids using PvuII
- Reaction mixture composition:
1 μl purified plasmid DNA product 0.5 μl PvuII (Fermentas) 2 μl Buffer Green (Fermentas) 16.5 μl MQ water
- After the digest each plasmid solution was loaded into the 1% agarose gel
verification of the
restriction patterns
restriction patterns
Electrophoresis condition:
voltage - 70V
time - 30 min
Comment:
The restriction patterns indicate that all isolated plasmids are empty
Testing different E. coli strains regarding [http://partsregistry.org/Part:BBa_R0010 lacI] and [http://partsregistry.org/Part:BBa_R0080 AraC] repressors
Franek
Tasks:
- Alkaline lysis of bacterial cultures to obtain [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] device
- Transformation of Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a competent cells with [http://partsregistry.org/Part:BBa_K177024 BBa_K177024]
Methods:
- 2 test tubes with 5ml LB and ampicillin were first inoculated with colonies containing [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] on [http://partsregistry.org/Part:pSB1A2 pSB1A2] plasmid. The cultures were incubated overnight at 37°C. Alkaline lysis was performed with [http://aabiot.com/products/dna_purification/plasmid_dna/plasmid_mini/protocol_plasmid_mini.pdf PlasmidMini set by A&A Biotechnology]. The pellet from 5 ml of bacteria was used.
- Chemocompetent E. coli cells from Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a strains were transformed according to our standard procedure with [http://partsregistry.org/Part:BBa_K177024 BBa_K177024]
Results:
- Will be determined by cell colonies presence on plates
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