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Revision as of 20:19, 14 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
2
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.
