Recipes for construction
| Primer 1 | 50 μL |
| Primer 2 | 50 μL |
| dNTP | 50 μL |
| Buffer | 100 μL |
| MgCl2 | 100 μL |
| dH2O | 600 μL |
PCR mixture
| Component | For 1 reaction (V=10µl) | For 1000 reactionsL |
| Forward primer | 0,5 μL | 50 μL |
| Reverse primer | 0,5 μL | 50 μL |
| Master mix k0171 | 5 µl | 500 μL |
| dH2O | 4 μL | 400 μL |
Restriction mixture
| DNA | 20 μL |
| buffer Tango | 2,5 μL |
| restriction enzyme 1 | 1 μL |
| Restriction enzyme 2 | 1 μL |
Ligation mixture
| Component | Volume (µl) |
| insert | 6 μL |
| vector | 2 μL |
| T4 DNA ligase | 1 μL |
| T4 DNA ligase buffer | 1 μL |
Incubate over night at 16°C or 2 hours at room temperature.
Ligation mixture for pJET blunt vector
| Gene | 7 µl |
| Vector | 1 μL |
| T4 DNA ligase buffer | 1 μL |
| T4 DNA ligase | 1 μL |
SDS-PAGE gel (12,5 %)
| Component | Separation gel (V= 10ml) | Stacking gel (V= 5ml) |
| dH2O | 4, 27 ml (5x854 µL) | 3, 08 ml (4x769 µL) |
| separating gel buffer | / | 1, 25 ml (2x625 µL) |
| stacking gel buffer (pH= 6,8) | / | 1, 25 ml (2x625 µL) |
| acrylamide bisacrylamide (37:1) | 3,13 ml (4x783 µL) | 0, 625 ml (625 µL) |
| 10% SDS | 100 µL | 50 µL |
| TEMED | 15 µL | 7,5 μL |
| 15 % APS | 30 μL | 15 μL |
| Component | For 1 litre | for 500 ml |
| Bacto-tryptone | 9 g | 4,5 g |
| Yeast Extract | 4,5 g | 2,25 g |
| Sodium chloride | 4,5 g | 2,25 g |
o Broth should be autoclaved (sterilization)
o Broth should be stored at room temparature until opened and then must be stored at 4°C
o It can also be re-autoclaved, but this is not recommended if:
- • If there is contamination
growth, autoclaving will kill
this but will not remove any
toxic waste products which
have been produced.
- • Autoclaving results in some
evaporation which will
increase the concentration
of the broth.
- • Once antibiotics have been
added it should not be re-
autoclaved.
Mixture:
| Component | For 1 litre | for 500 ml |
| Bacto-tryptone | 9 g | 4,5 g |
| Yeast Extract | 4,5 g | 2,25 g |
| Sodium chloride | 4,5 g | 2,25 g |
| Bacto Agar | 1,5 g | 0,75 g |
o Once autoclaved the LB AGAR can be used as soon at it has cooled to ~55oC, or it may be stored at room temperature and microwaved when required.
o When microwaving LB AGAR only use medium heat to prevent super heating and uneven melting which can adversely affect the smooth finish of plates.
Colouring fluid:
o 20% acetic acid and Comassi e Brilliant Blue dye must be mixed 1:1 (20µL:20µL).
Fluid for discoloration:
o 30% ethanol, 10% acetic acid
Comassie Brlliant Blue
< o Mix 1 g of comassie Brilliant Blue powder with 200 ml ethanol 300 ml H2O
Separating Buffer
o 1,5M Tris/HCl, pH= 8,8
Stacking Buffer
o 0,5 M Tris/HCl, pH= 6,8
10xSDS Buffer
o 30g Tris, 144g Glycine, 10 g SDS (sodium dodecyl sulfide)
Transferring the protein from the gel to the membrane ( Western blot)
Preparation of western transfer buffer:
| Tris | 3 g |
| Glycine | 14,4 g |
| Methanol | 200 μL |
| H2O | 800 μL |
• Activate the membrane by covering in methanol and rince the membrane, sponges, filters and gel with transfer buffer.
• Prepare the stack as follows (loading on the anode): sponge, 3 filters, gel, membrane, 3 filters, sponge. Make sure no air bubbles are traped in the stack.
• Place the cassette in the transfer tank togheter with an ice pack. Fill the tank with transfer buffer to the appropriate line.
• Run at 200 mA for 1,5 hours.
• Store the membrane in 5% milk for 1 hour at room temperature.
Preparation of PBS buffer:
| NaCl | 5,8 g |
| Na2HPO4 | 8,9 g |
•20mM Hepes
•500 mM NaCl
•20mM imidazole
•fill with H2O till 250 ml
•pH=7, 5
Elution buffer
•20mM hepes
•500mM naCl
•250 mM imidazole
•fill with H2O till 250 ml
•pH=7,5
Recipes for testing
•The procedure for bioreactor preparation2 grams of glucose are added into 500 ml bioreactor. Glucose serves as nutrition for the mixed culture, present in the inoculum. In the beaker, 300 g of inoculum and 100 g of deionized water are weighted for each bioreactor. 12 reactors are prepared simultaneously. Then, pH of the solution in each bioreactor is set on 5.7 with 2 molar hydrogen chloride. When setting the appropriate pH, the solution is mixed with the mixer in order to give a homogenous solution. 400g of the solution is weighted in the bioreactor and purged with nitrogen for 10 minutes without being closed. Set a mechanic mixer with one pipe, leading directly into inoculum, and one for measuring the products in bioreactors. The bioreactors were then purged again, this time with argon for 5 minutes. Argon is used because it ensures anaerobic environment alike nitrogen, and because it does not form compounds with the substances in inoculum. The protocol is the same as above mentioned with some modifications: two bioreactors were ‘blind’ without any added glucose that serves as control bioreactors and 6 bioreactors contained 2 g of glucose per 400 g of solution (inoculum + deionized water). 200 ml of sample with adjusted pH at 5.7 got frozen.
Preparation of samples for HLPC measurementsThe syringe is placed on the neck of the plastic tube of bioreactor and then you open the valve of the tube and take the sample (approximately 2 ml). Next, close the valve and transmit the sample into microcentrifuge tubes that had to be marked with the time and date of sampling. The volume of the taken sample is constantly recorded to prevent: lest the removed solution from affecting the final production. Before proceeding on, the plastic tube has to be purged with argon, to prevent inoculum from staying inside the tube and preventing the sampling in advance. Be careful; use only the amount of argon that corresponds the volume of the plastic tube so no argon is purged in the sample.
Transmit the sample in the microcentrifuge tubes and weigh them. One microcentrifuge tube with the water, that weights the same as a sampling microcentrifuge tube, must be prepared for centrifugation. In the centrifuge, each microcetrifuge tube has to have the complementary tube that weights the same and it is placed in the hole opposite the hole with the sample in it. Microfuges tubes are centrifuged for 20 minutes at 10 000 rotations per minute with intent to remove solid substances. 0.3 ml of the supernatant is then diluted with 2.7 ml of distilled water and filtrated approximately 1 ml – 1.5 ml into vials.
Prepare 2 vials with MQ for equilibration. All samples must be filtrates and checked if they are suitable for HPLC grade. Conditions for HLPC measurements:
•HI-Plex colon (Agilent Techonologies, 7.7x 300mm, 8 µm)
•DAD detector
•Temperature = 50°C
•0.01 M H2SO4
•Flux: 0.6 ml / min
•Volume: 20 µl
Analysis of butyric acid occurred on DAD detector at 210 nm.
Preparation of standard ENDO medium: For 100 ml of ENDO medium (BUJON) with lactose and antibiotic:
•1,00 g of meat peptone
•1,00 g of lactose
•0,25 g of sodium sulfite
•0,35 g of dipotassium hydrogen phosphate
Preparation of peptone water (250 ml): •1,50 g of peptone
•1,75 g of sodium chloride
Add MQ to the mark labelling 250 ml.
The chemical composition of 72 different media
SLIKA TABELE GOJIŠČ!!!!!!!!!!
Instructions for media preparation
At the begging, prepare yourself a solution of HCl and solution of NaOH in the beaker, which serve you as the pH regulators. Prepare the beaker with distilled water for deposition of pH electrode.
For HCl solution, prepare 20 ml of strong HCl and dilute it in 100 ml of distilled water. For NaOH solution, prepare 40 g of NaOH and dilute it in 200 ml of distilled water. Use glasses and be careful; solution can boil!
Media G1-G4 prepare according to the upper table in the Falcon cetrifugate tubes.
Butyric acid is measured with pipette:
•0,12 g = 126 µL
•0,080 g = 84 µL
•0,040 g = 42 µL
Glycerol, lactose, NaCl and yeast extract are weighed.
Other media prepare in the 2000 ml flask (which is put on the balance and place TARE in advance) following chemicals:
•20 g of meat peptone
•7 g of dipotassium hydrogen phosphate
•5 g of sodium sulfite
All chemicals are weighted with analytic balance in the weighing boats, which must not be wet during the weighting. Add a few milliliters of MQ to dissolve the chemicals and then fill the flask to the begging of narrow neck. First prepare the flask with pH=5, then repeat the procedure for pH = 6, pH=7, pH=8.
Media G5-G68 are prepared following these instructions:
•Put the beaker with the empty Falcon centrifuge tube on the balance and press TARE
•Weigh 35 g of solution from the flask and pour the chemicals in it. Always wash the weighing boats with water and pour the water in the Falcon centrifuge tube
•Add the media from the flask up to 39 g and regulate the pH value with appropriate NaOH or HCl solution
•Place the tube on the Vortex machine for 20 seconds
•Check the pH value again and if it is not suitable, regulate it again!
•Fill the Falcon centrifugate tube up to 40,00 g and close it.
Inoculation of genetically modified E.coli:
Bacteria were inoculated on the new LB plate in laminar:
•One colony is taken from the previous plate with inoculation loop
•Gently draw a new plate with inoculation loop (6-times left and right, one down and another left to right)
•Wrap the LB plate with the parafilm
•Date and name of the bacteria were written on each sample and medium were put in the incubator set on 37°C.
Instructions for an autoclave:
Autoclave is filled with laboratory equipment for disengagement, closed with the lid and the lid is tighten with screws. The level of water is checked; if not in the blue area, you open the valve and add distilled water through the funnel and close the valve. Then, close the steam conduit and turn on the autoclave with the switch on the wall so that the red light lits. When the pressure reaches the value of 1 bar, the green light turns on and the autoclaving begins. Gently open the steam conduit. After the autoclaving is over, the autoclave must be turn off, the filtration of air is turned on and the steam conduit is completely opened. Wait until the autoclave is completely cooled down to open the lid.
Preparation of physiological solution (500ml 0,9 % NaCl )
4,5 g NaCl is diluted in 500 ml of water in the flask and transmitted in the laboratory bottle with blue lid, which must be howled only twice for the autoclave.
The sterilization of antibiotic
Engulf antibiotic with the injection from microcentrifugate tube, place the sterile filter on it and pour antibiotic in the new microcentrifugate tube. Mark the tube with appropriate signification.
Biosafety cabinet
First you have to turn on the cabinet, set the eager conditions and then disinfect it at least 10 minutes before you are starting to work. The work must run from the sterile to the infected side and never opposite! When you finish, disinfect all objects before you remove them from the biosafety cabinet. Turn off the biosafety cabinet.
The analysis of the waste glycerol
•CHNS analysis
•Karl-Fitcher titration
The sample is diluted with water by the factor 100 and filtered through the filter into a vial. Then, standard HPLC analysis is done.
Settings for the PIPETIRNI ROBOT
Change adapter-LAN-properties-PV4+ kode (IP address)
IP addresses:
192.168.65.67
192.168.65.14
255.255.255.0
193.2.1.66
192.168.65.254
Second icon: ‘PING’ + ‘address + 137’ → ‘send 4, receive 4’
The method of butan-1-ol insulation
•The water from bio filter is used (18,2 Ω/cm)
•5% solution of butan-1-ol is prepared
•10 ml is transferred in the 100 ml flask and diluted to give 0,5% solution
•10 ml is transferred in the second 100 ml flask and diluted to give 0,05% solution
•1% solution of butan-1-ol is prepared
•1,1 ml of solution is taken out of each flask and poured into 3 microcentrifuge tubes
•Then, cetrifugate the tubes:
1. At 5000 rpm for 1 minute
2. At 5000 rpm for 5 minutes
3. At 5000 rpm for 10 minutes
4. At 10000 rpm for 1 minute
5. At 10000 rpm for 5 minutes
6. At 10000 rpm for 10 minutes
Before the centrifugation process prepare:
•100 ml of 0,25 M potassium dichromate
•100 ml of 0,1 M silver nitrate
•100 ml of 6 M sulphur (VI) acid
•After that, prepare 4 centrifuge tubes following the next protocol:
•5 ml of potassium dichromate solution in each
•1 drop of silver nitrate
•Mix immediately!
•Add 5 ml of H2SO4
•Mix again
•Add 1 ml of 5% solution of butan-1-ol in the first centrifuge tube, 1 ml of 0,5% solution of butan-1-ol in the second, 1 ml of 0,05% solution of butan-1-ol in the third and 1 ml of 1% solution of butan-1-ol in the forth. All centrifuge tubes are mixed on the Vortex machine and left still for at least 5 minutes to ensure that the reaction occurs. 29 ml of distilled water is added in each centrifuge tube and they are placed on the Vortex machine again. At the end, measure the amount of absorbance at 560 nm with spectrophotometer.
The centrifugation process:
•100 µl is transferred from the microcetrifuge tubes to new microcetrifuge tubes and 900 µl of MQ is added.
•5 ml of potassium dichromate solution in each
•1 drop of silver nitrate
•ix immediately!
•Add 5 ml of H2SO4
•Mix again
The standardized protocol is repeated for all cycles, all measurements are repeated three times (18 cycles).
The preparation of biofilm Escherichia coli
Our team prepared 100 ml of ENDO medium (BUJON) with lactose and antibiotic:
· 1,00 g of meat peptone
· 1,00 g of lactose
· 0,25 g of sodium sulfite
· 0,35 g of dipotassium hydrogen phosphate
We also prepared 250 ml of peptone water:
· 1,50 g of peptone
· 1,75 g of sodium chloride
Add MQ up to the labelled mark in both solutions. Both media are placed in the autoclave simultaneously with the holders for 50ml biofilm. When all items are sterilized, they are placed directly in the laminar. At the moment when the temperature of the both media is 60°C the antibiotic was added (1 µl on every 1ml of medium), which is previously filtered through the sterilized filter (0,2 µm pore). After the ENDO medium reaches a room temperature, the bacteria, growing on the LBC medium in the incubator on 37°C, are inoculated. Then, the conical flask is put on the shaker (100 rmp) for 12 hours at 37°C and is used for the transmission of the medium into sterilized 26-well plate the next day. In each well was added with 100 µl of diluted solution of bacteria in peptone water, diluted in the ratio 1:100. The sample was incubated for 3 days at 37°C without shaking the well plate.