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Team:Warsaw/Calendar-Main/16 July 2009
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Cloning of the mgtc promoter into the pKSII+ plasmid
Kamil
Tasks:
- Transformant selection
Methods:
- White colonies were picked up and transferred to a new petri dish.
- Liquid cultures were established along the way for plasmid purification.
- Both the dish and the liquid cultures were incubated at 37°C overnight.
Cloning of p53 coding sequence
Marcin
Task:
- Transformation of chemocompetent E. coli strain DH5α
Methods:
- Ligation reaction was stopped via thermal inactivation in 80°C for 20 minutes
- Detailed protocol of ligation is described here. The only modification is usage of half volume of ligation mixture prepared 15.07.09
Assembly of endosomal detection operon
Marcin
Task:
- Restriction digest of biobricks
Comment:
Due to obtain set of biobricks which each of them contain RBS and particular coding sequence some of biobricks were digested:
Methods:
- Digest of BBa_B0032 using SpeI and PstI
- Reaction mixture composition: 10 μl purified plasmid DNA product, 0.5 μl SpeI (Fermentas),1 μl PstI (Fermentas), 5 μl Buffer Tango (Fermentas), 34 μl MQ water
- Digest of other biobricks using PstI and XbaI
- Reaction mixture composition: 10 μl purified plasmid DNA product, 1 μl XbaI (Fermentas),1 μl PstI (Fermentas), 5 μl Buffer Tango (Fermentas), 34 μl MQ water
- Both reaction were performed overnight (~12 hours) and both of them were subsequently inactivated via heating in 80°C for 20 minutes
Construction of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 K177012] operon1_part2
Ania
Tasks:
- Set up a liquid culture from transformants containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032 - RBS.3] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012 - lacI repressor] on the pSB1A2 ampicillin resistant plasmid.
- Alkaline lysis of bacterial cultures to obtain plasmid.
- Digest of the ligated [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032 - RBS.3] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012 - lacI repressor] on the pSB1A2 ampicillin resistant plasmid with EcoRI and PstI to confirm ligation.
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] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025] devices
- Transformation of Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a competent cells with [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025]
Methods:
- 2 test tubes with 5ml LB and ampicillin were first inoculated with colonies containing [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] and [http://partsregistry.org/Part:BBa_K177025 BBa_K177025] on [http://partsregistry.org/Part:pSB1A2 pSB1A2] plasmid. The cultures were incubated overnight at 37°C. Alkaline lysis was performed on both cultures, according to our standard procedure. The pellet from 5 ml of bacteria was used.
- Chemocompetent E. coli cells from Top10/ BL/ GM/ V1011/ V1012/ TopF'/ DH5a strains (prepered earlier by Kuba) were transformed according to our standard procedure with either [http://partsregistry.org/Part:BBa_K177024 BBa_K177024] or [http://partsregistry.org/Part:BBa_K177025 BBa_K177025]
Results:
- Will be determined by cell colonies presence on plates
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