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Team:Warsaw/Calendar-Main/24 July 2009
From 2009.igem.org
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Cloning of the mgtc promoter into the pKSII+ plasmid
Kamil
Tasks:
- Plasmid purification
- Control digest
Methods:
- Despite the fact that all transferred colonies turned more or less blue the purification was carried out using the Plasmid Mini kit (A&A Biotechnology) according to the manufacturers protocol.
- The digest mix was prepared as fallows: 10μl of purified plasmid, 2μl Tango buffer (Fermentas), 1μl SpeI enzyme, 1μl XbaI enzyme, the solution was topped up with H2O to the final volume of 20μl.
- The digest was incubated for 3h at 37°C.
- The results were visualised with gel electrophoresis on 1% agarose gel.
Results:
- Gel Electrophoresis:
Sukcesy
Nigdy mi nie odmówiły
Te, które mi się śniły.
(Jan Sztaudynger)
Assembly of endosomal detection operon
Marcin
Task 1:
- Isolate the plasmids from bacterial cultures and verify the effectivity of the isolation via gel electrophoresis
- Plasmids weren isolated using the A&A plasmid mini kit. Detailed procedure of the isolation is described here
- After the isolation 1 ul of each plasmid solution was diluted to 10 ul and loaded into the 1% agarose gel
- Electrophoresis condition:
Methods:
voltage - 70V
time - 30 min
Task 2:
- Digest of isolate plasmids with ligated biobricks to verify the success of ligation
Methods:
- Digest of isolate plasmids using EcoRI and PstI
- Reaction mixture composition: 0.5 μl purified plasmid DNA product, 0.5 μl EcoRI (Fermentas),0.5 μl PstI (Fermentas), 2 μl Buffer Tango (Fermentas), 16.5 μl MQ water
- The reaction was performed three hours and it was subsequently inactivated via heating in 80°C for 20 minutes.
- In the next step reaction mixtures were loaded into the agarose gel to analize restriction pattern of the plasmids
Comment:
The procedure of digest was incorrect due to use Tango Buffer - EcoRI need specific buffer and it may do not work correctly in this buffer. It is obligated to repeat the procedure using modified set on restriction enzymes.
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