Team:elan vital korea/Protocol








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 be mixed completely.


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.

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).



To The Top


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.



LB Agar Plates and Addition of Antibiotics

1.
We have used LB (solidified lysogeny broth), rich growth medium for E.coli, in our experiments almost all the time.

2.
All the needed chemicals including yeast extract must be prepared beforehand.

3.
Autoclave must be available.

4.
Growth on LB Agar plates provides colonies originating from one single bacteria cell.

5.
Just before pouring the solution into petri dishes, an antibiotic can be added for resistance selection.
We followed the normal working concentrations such as:
- chloramphenicol: 25 μg/mL
(Chloramphenicol stock is dissolved in ethanol)
In case of using ampicillin: 100 μg/mL
- normal stock concentrations:1000-fold

6. Material to make LB plates:
NaCl (Sodium Chloride)
Bacto™ tryptone
yeast extract
Bacto™ agar
ddH2O (triple distilled water)
1000x chloramphenicol or ampicillin

To The Top

7. Process
We usually make 1liter bottle for LB Agar
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

1. 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. 2. Materials: NaCl, Trypton, Yeast Extract, and ddH2O (triple distilled water) 3. Equipment: autoclave, electronic scale. 4. Process 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



Overnight Cultures with Antibiotics