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Revision as of 17:46, 17 September 2015
WETLAB
-Protocol-
PROTOCOL
We conducted our experiments by following the protocols below. As an official procedure, lab workers should understand the lab experiment
assigned to them along with safety procedures before starting lab work. The protocols are arranged according to the order of experiments we followed.
Protocols to handle enzymes.
1
Enzymes used in our project, such as AHL, must be stored
in low temperature.The enzymes must be stored in the
freezer when they are not used, and must be put on ice when
taking them out of the freezer for an experiment.
2
Enzymes should be added last to the solution, because
enzymes are sensitive to inactivation by pH and
ionic conditions that deviate from their storage and reaction
buffers. After adding enzymes, the mixed solution should
Protocols to store materials and maintain
usage history of each material.
1
Reporter cell, test cell and competent cell (Top 10 invitrogen)
must be kept at 4°C and frequently used
enzymes, reagents, DNA plasmids should be kept at −20°C
in the freezer..
2
We use AHL as protein enzymes. AHL must be kept at lower
temperature (4°C or lower) for the recurring use.
AHL can be destroyed easily when it is stored and/or handled
in improper temperature.
To The Top
3
We use triple distilled water (or
DDH2O) to make LB broth.
Triple distilled water is kept at lab
temperature(around 18 °C or lower).
4
Other materials such as yeast and
NaCl are stored and maintained
under the responsibility of
Gachon Molecular Biology Lab.
5
We have to record the history of each
material, including if
plasmids/reporter cell/ test cell/ AHL have
been frozen and if so,
when it is used.
Process
1
200 mL LB prepared fresh,
non-autoclaved
2
3 g agar
3
Shake until all solids
are dissolved
4
Autoclave for 20 min within 2 hr
5
Keep it cool until it reaches
around 40-50 °C
6
Add 200 μL of 1000x chloramphenicol
and gently stir it. Be careful not
to shake the bottle too long/hard so that
bubbles are created.
7
Pour into empty petri dishes just enough
to cover the surface (~20 mL per plate).
In case that bubbles are in
the plate, heat the plate surface carefully
with a burner only until the bubbles are
burst but the solution is heated.
8
Leave the plates at room temperature
around one hour until it is solidified.
9
Solidified plates should be turned upside
down for a few hours at room temperature,
then stored at 4°C.
LB Medium
We used LB medium almost every day, so we have prepared LB medium many times. To prevent contamination, we only used LB medium made within three days.
NaCl, Trypton, Yeast Extract, and ddH2O (triple distilled water)
autoclave, electronic scale.
We usually make 200 liter LB bottle
1) 2 g of NaCl to a final concentration of 0.17 M
2) 2g of 1%(w/v) Bacto™ tryptone
3) 1g of 0.5% (w/v) yeast extract
4) ddH2O to 200 mL
5) Autoclave for 20 min within 2 hours
6) Keep at room temperature
We have conducted overnight culture for a single bacterial strain which process needs a plate or medium with single colonies and LB containing chloramphenicol.
chloramphenicol or ampicillin should not be added to LB before autoclaving the LB or after subsequent cooling to room temperature for storage because antibiotics are not sufficiently stable. The solution must be added just before bacteria is added.
Material Needed chloramphenicol: 25 μg/mL
Normal stock concentrations: 1000-fold higher
In case of using ampicillin: 100 μg/mL
4. Process
1) Quickly burn the neck of a bottle containing LB medium before pouring it out into a tube. Even the
slightest contamination of LB will be damaging.
2) Add chloramphenicol or ampicillin to give the appropriate concentration
3) Scoop one colony from the plate with a sterile micropipette tip
4) Immediately stick the tip into the tube containing the medium and chloramphenicol or ampicillin
5) Cover the tube loosely with aluminum foil by taping it on to allow exchange of oxygen for chemical maturation of the chromoprotein choromophores.
6) Incubate at 37°C with the shaking incubator overnight.
1
2 Materials
3 Equipment
4 Process
Overnight Cultures with Antibiotics
1
2 Materials
3 Equipment
4 Process