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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:
From left:
- GeneRuler DNA Ladder Mix #SM0333 (Fermentas)
- #1 Digest
- #2 Digest
- #3 Digest
- #4 Digest
- #5 Digest
NOTE: All of the samples consisted of just a linear plasmid.
Conclusions:
- A colony is blue for a reason.
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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
- Preparation of electrocompetent E.coli strain TOP10
- detailed ptotocol of preparation
- Prepare new bacterial culture - add 10 ml of previously prepared bacterial inoculum to 1 l of LB liquid medium.
- Incubate the culture in 37°C until the absorbance of the bacterial solution will be at least 0.5
- Cooled tne bacteria on ice for at least 25 minutes.
- Centrifuge the culture for 15 minutes at 4000g for 4°C minutes. The temperarure must be below 5°C.
- Carefully remove the supernatant and resuspend the bacteria in 1 l of cold water.
- Centrifuge the bacteria in the same manner as previously.
- Resuspend the bacteria in 0.5 l of cold water.
- Repeated the centrifuge procedure. Resuspend carefully last formed pellet in 10% solution of the glycerole
- Centrifuge the culture for 15 minutes at 4000g for 4°C minutes. The temperarure must be below 5°C
- Resuspent the bacterial cells in 3 ml of 10% solution of the glycerole and prepare the aliquots of the bacteria which all contains about 200 μl of the solution
- Freeze the bacteria in liquid nitrogen.
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.
Preparation of the electrocompetent bacteria
Marcin
Task 1:
Methods:
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