Team:elan vital korea/Protocol
WETLAB
-Protocol-
Protocols
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.
How to handle reagents.
1.
Reagents used in our project, such as restriction reagents, must be stored in low temperature. The reagents 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.
Reagents should be added last to the solution, because reagents are sensitive to inactivation by pH and ionic conditions that deviate from their storage and reaction buffers. After adding reagents, 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 reagents, reagents, DNA plasmids should be kept at −20°C in the freezer.
2.
We use triple distilled water (or DDH2O) to make LB broth. Triple distilled water is kept at lab temperature (around 18 °C or lower).
3.
Other materials such as yeast and NaCl are stored and maintained under the responsibility of Gachon Molecular Biology Lab.
4.
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.
To The Top
LB Medium
1. To prevent contamination, we only used LB medium made within three days.
2. Materials: Sodium Chloride (LB Media, Sigma), Trypton(LB Media, Sigma), Yeast Extract(LB Media Sigma), ddH2O (triple distilled water)
3. Equipment: autoclave, electronic scale.
4. Protocol For 200mL LB bottle
1) 2 g of Sodium Chloride 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
LB Agar Plates and Addition of Antibiotics
1. We have used LB (solidified lysogeny broth), rich growth medium for E.coli, in our experiments.
2. 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
3. Material to make LB plates: Sodium Chloride (LB Media, Sigma) Bacto™ tryptone (LB Media, Sigma) yeast extract (LB Media, Sigma) Bacto™ agar (LB Media, Sigma) ddH2O (triple distilled water) 1000x chloramphenicol or ampicillin
4. LB agar preparation protocol 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.
Overnight Cultures with Antibiotics
1. We have conducted overnight culture for a single bacterial strain which process needs a plate or medium with single colonies and LB containing chloramphenicol.
2. Material Needed chloramphenicol: 25 μg/mL Normal stock concentrations: 1000-fold higher In case of using ampicillin: 100 μg/mL
3. Protocol
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) Incubate at 37°C with the shaking incubator overnight.
Agarose Gel Electrophoresis
1. Agarose gel electrophoresis is used for separation and analysis of larger (>100 bases in length) nucleic acids under non-denaturing conditions.
2. Analysis requires that the gel contains a DNA stain visible under UV light. Since the stain interacts with nucleic acids and is therefore potentially mutagenic, always wear nitrile gloves when working with agarose gels.
3. Use protective glasses when using the UV light box.
4. Material Needed
Agarose
1x TBE
Sybr®Safe
Loading dye mix
DNA ladder size marker
DNA samples
5. Protocol:
1) The gel tray must be on a level surface.
2) Insert the comb into the gel tray at one end ~1 cm from the edge.
3) For a 1% 150 mL agarose gel, weigh 1.5 g of agarose in a 500 mL conical flask.
4) Add 150 mL 1x TBE buffer.
5) To dissolve the agarose in the buffer, swirl to mix and microwave for a few minutes taking care not to boil the solution out of the flask.
6) Remove the flask occasionally and check whether the agarose has dissolved completely.
7) Let the agarose solution cool down.
8) Once the solution is touchable, add the DNA stain.
9) Check the stock concentration as the working concentration for ethidium bromide is 0.5 μg/mL while for Sybr®Safe it is simply 1x.
10) Pour the gel solution into the gel tray.
11) Remove any air bubbles with a pipette tip.
12) Put in comb.
13) The gel will solidify while cooling down to room temperature, which usually takes about 30 min.
14) Running the gel by the following procedure
a. Release the gel tray from the tape or casting stand.
b. Place the gel tray into the buffer chamber and remove the comb carefully
c. Add 1x TBE buffer until the gel is completely covered.
d. Take the DNA sample (~0.2 μg) and mix with loading dye.
e. Load the size marker mixed in 1x loading dye (~6 μL final volume) into a middle well.
f. Load the samples into the other wells while writing down which lanes have which samples.
g. Put the lid onto the buffer chamber and connect it to the power supply.
h. Run the gel at 100 V for 30–60 min. Neither of the two dyes should be run off the gel.
i. Stop the run and bring the gel to a UV table to visualize the gel bands.
j. Take a picture of the gel.
Gel Extraction
QIAquick®Gel Extraction Kit
Notes before starting
1. This protocol is for the purification of up to 10μg DNA (70bp to 10kb).
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every tube every 2-3 min to help dissolve gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the color of the mixture is orange or violet, add 10μl 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow.
8. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
On each chemical container, there is a label that specifies the potential danger of the substance for humans and/or the environment.
When you are working to generate new organisms by molecular methods, you are regulated in most countries
CDC, Atlanta. Biosafety in Microbiological and Biomedical Laboratories.
WHO, Geneva. Laboratory Biosafety Manual, 2004.
ECDC, Directive 2000/54/ec of the European parliament and of the Council of 18 September 2000 on the protection of workers from risks related to exposure to biological agents at work (seventh individual directive within the meaning of Article 16(1) of Directive 89/391/EEC.
Institutional Bio-safety Committee, under the ministry of Health and Welfare, Korea Center for Disease Control & Prevention.
When working with microorganisms such as bacteria and viruses, there are four BioSafety Levels (BSL) numbered BSL1–4.
involving well-characterized agents not known to consistently cause disease in healthy adult humans and of minimal potential hazard to laboratory personnel and the environment.
bacteria and viruses including canine hepatitis, non-pathogenic Escherichia coli, as well as cell cultures and non-infectious bacteria.
The laboratory is not necessarily separated from the general traffic patterns in the building.
Conducted on open bench tops using standard microbiological practices.
Laboratory personnel have specific training in the procedures conducted in the laboratory.
Supervised by a scientist with proper training.
Contaminated materials are left in open waste receptacles.
Similar to Biosafety Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment.
Includes various bacteria and viruses that cause only mild disease to humans, or are difficult to contract via aerosol in a lab setting such as C. difficile, most Chalmydiae, hepatitis A,B, and C, orthopoxviruses, influenza A, Lyme disease, Salmonella, mumps, measles, scapie, MRSA, and VRSA.
similar to Biosafety Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. It includes various bacteria and viruses that cause only mild disease to humans, or are difficult to contract via aerosol in a lab setting.
Laboratory personnel have specific training in handling pathogenic agents,
Must be directed by scientists with advanced training.
Extreme precautions are taken with contaminated sharp items.
In case that infectious aerosols or splashes may be created, specified procedures should be conducted in biological safety cabinets or other physical containment equipment.
Indigenous or exotic agents which may cause serious or potentially lethal disease after inhalation.
Includes various bacteria, parasites and viruses that can cause severe to fatal disease in humans but for which treatments exist, such as Yersinia pestis, SARS coronavirus, Brucella, yellow fever virus.
Laboratory personnel have specific training in handling pathogenic and potentially lethal agents,
Supervised by competent scientists who are experienced in working with these agents.
All procedures involving the manipulation of infectious materials are conducted within biological safety cabinets, specially designed hoods, or other physical containment devices.
The laboratory personals should wear appropriate personal protective clothing and equipment.
An acceptable level of safety for the conduct of routine procedures, may be achieved in a biosafety level 2 facility, providing the filtered exhaust air from the laboratory room is discharged to the outdoors.
The ventilation to the laboratory is balanced to provide directional airflow into the room.
Access to the laboratory is restricted when work is in progress and the recommended Standard Microbiological Practices, Special Practices, and Safety Equipment for Biosafety Level 3 are rigorously followed.
Limited access only for authorized persons can work in the facilities. Not allowed for people who have weak immune system.
When dealing with biological hazards at this level, the use of a positive pressure personnel suit, with a segregated mandatory.
All works should be supervised by qualified scientists who are trained and experienced in working with these agents.
The entrance and exit of a level 4 bio-lab will contain multiple showers, a vacuum room, an ultraviolet light room, and other safety precautions designated to destroy all traces of the biohazard.
Multiple airlocks are employed and are electronically secured to prevent both doors from opening at the same time.
All air and water service going to and coming from a biosafety level 4 will undergo similar decontamination procedures to eliminate the possibility of an accidental release.
Members of the laboratory staff shall have specific and thorough training in handling extremely hazardous infectious agents, and they shall understand the primary and secondary containment functions of the standard and special practices, the containment equipment, and the laboratory design characteristics.
2. The yellow color of buffer QG indicates a pH ≤ 7.5. DNA adsorption to the membrane is only efficient at pH ≤ 7.5.
3. Add ethanol (96%100%) to Buffer PE before use (see bottle label for volume).
4. Isopropanol (100%) and a heating block or water bath at 50°C are required.
5. All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a convetional table-top microcentrifuge.
6. Symbosl: ● centrifuge processing; ▲ vacuum processing.
1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg gel ~ 100μl). The maximum amount of gel per spin column is 400mg. For >2% agarose gels, add 6 volumes Buffer QG.
4. Add 1 volume isopropanol to the sample and mix.
5. Place a QIAquick spin column in ● a provided 2ml collection tube or into ▲ a vacuum amnifold. To bind DNA, aply the sample to the QIAquick column and ● centrifuge for 1 min or ▲ apply vaccum to the manifold untill all the
samples QIAquick column back into the same tube. For example volumes of 0> 800μl, load and spin/apply vacuum again.
6. If the DNA will subsequently be used for sequenceing, in vitro transcruption, or microinjection, add 500μl Buffer QG to the QIAquick column and ● centrifuge for 1 min or ▲ apply vaccum. ● Discard flow through and place the
QIAquick column back into the same tube.
7. To wash, add 750μl Bufick column and fer PE to QIAquickcolumn and ●
centrifuge for 1 min or ▲ apply vacuum. ●Discard flow-through and place the
QIAquick column back into the same tube.
9. To elute DNA, add 50μl Buffer EB (10mM Tris•Cl, pH 8.5) or water to the
center of the QIAquick memberane and centrifugethe colum for 1 min. For
increased DNA concentration, add 30μl Buffer EB to the center of the
QIAquick membrance, let the column stand for 1 min, and then centrifuge for
1 min. After the addition of Buffer EB to the QIAquick membrance, increasing
the incubation time to up to 4 min can increase the yield of purified DNA.
10. If the Purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye
to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
CHEMICALS
Chemicals should be handled cautiously with gloves, both for your safety and for decreasing the contamination risk.
Always wear a lab coat and shoes as additional protection. Read the signs on the chemical container and the Material Safety Data Sheet
(MSDS; available online) for further direction.
BIO- SAFETY AND DISPOSAL
according to international biosafety guidelines:
1
http://www.cdc.gov/biosafety/publications/bmbl5/bmbl5_sect_iv.pdf
2
http://www.who.int/csr/resources/publications/ biosafety/Biosafety7.pdf
3
http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2000:262:0021:0045:EN:PDF
4
http://biosafety.cdc.go.kr
Biosafety level is the level of the biocontainment precautions required to isolate dangerous biological agents in an enclosed
The levels of containment range from the lowest biosafety level 1 to the highest at level 4, and Ministry of Health and Welfare and Korea Centers
for Disease Control and Prevention have specified these levels facility.
Biocontainment can be classified by the relative danger to the surrounding environment as biological
safety levels. As of 2006, there are four safety levels. These are called BSL1 through BSL4
BSL 1
1
2
3
4
5
6
7
BSL 2
1
2
3
4
5
6
7
BSL 3
1
2
3
4
5
6
7
8
9
BSL 4
1
2
3
4
5
6
7