Team:UiOslo Norway/Experiments

UiOslo

Experimenting is fun!

Gel Extraction

MATERIALS:

Scalpel blade
Thermo Scientific GeneJET gel extractionkit containing:

100-100ul Binding Buffer
GeneJET Purificatin column
700ul Wash Buffer (Pre-mixed with the appropriate amount of 99% ethanol)

1.5mL Eppendorf tube
51ul Reverse osmosis water

EQUIPMENT:

Thermocycler, heating block, or water bath
Desktop Centrifuge
Nanodrop spectrophotometer

PROTOCOL:

- Excise the band of interest from the agarose gel

- Add 1μL Binding Buffer for every 1mg of agarose gel

- Incubate at 50°C – 60°C for 10 minutes, inverting every 2 minutes

(Incubate for longer and invert every minute if gel hasn't fully dissolved)

- Transfer gel solution to spin column

- Centrifuge at 12 000g for 1 minute, discard flowthrough

- Add wash buffer to the column

- Centrifuge a second time at 12 000g for 1 minute, discard flowthrough

- Centrifuge a third time at 12 000g for 1 minute, discard flowthrough

- Place the column into a new Eppendorf tube

- Add 50μL reverse osmosis water to the column

- Centrifuge a fourth time at 12 000g for 1 minute, discard flowthrough

- Initialize and blank Nanodrop spectrophotometer with 1μL reverse osmosis water

- Measure DNA concentration with Nanodrop spectrophotometer

- Store purified DNA at -20°C

REFERENCE: From Thermo Scientific GeneJET Gel Extraction Kit protocol

DNA Extraction from Bacterial Cells

MATERIALS:

5mL LB media
5μL 100mg/mL ampicillin (Or an appropriate amount of another antibiotic)
50mL Falcon tube or similar container
Agar plate with colonies of interest
Tape
QIAprep Spin Miniprep Kit containing:

250μL Buffer P1
250μL Buffer P2
350μL Buffer N3
QIAprep Spin column
0.75mL Buffer PE

1.5 mL Eppendorf tube
51μL Reverse osmosis water

EQUIPMENT:

Gas flame or biological materials hood
Shaking Incubator
Desktop Centrifuge
Nanodrop spectrophotometer
-20°C freezer

PROTOCOL:

- Add ampicillin to LB media

- Inoculate media with the colony in the presence of a gas flame, biological materials hood or other sterile environment

- Incubate the culture for 16 hours at 37°C with 250 rpm shaking, ensure the container lid is not airtight such as by taping a loose lid on

- Centrifuge the culture for 10 minutes at 4000g and 4°C, discharge the supernatant

- Resuspend the culture in buffer P1 and transfer to 1.5 mL Eppendorf tube

- Add buffer P2, invert to mix

- Incubate for 4 minutes

- Add buffer N3 and invert to mix

- Centrifuge for 10 minutes at 12 000g and 4°C

- Pipette out the supernatant and apply to a QIAprep spin column

- Centrifuge for 1 minute at 12 000g and 4°C, discharge supernatant

- Add buffer PE

- Centrifuge a second time for 1 minute at 12 000g and 4°C, discharge supernatant

- Centrifuge a third time for 1 minute at 12 000g and 4°C, discharge supernatant

- Transfer column to an Eppendorf tube

- Add 50μL reverse osmosis water

- Centrifuge a fourth time for 1 minute at 12 000g and 4°C, discard column

- Initialize and blank Nanodrop spectrophotometer with 1μL reverse osmosis water

- Measure DNA concentration with Nanodrop spectrophotometer

- Store purified DNA at -20°C

Protocol from the QIAprep Spin Miniprep Kit

MATERIALS:

2.40g Agarose
300mL TAE Buffer
5μL GelRed Dye

EQUIPMENT:

Microwave (~600W)
Gel tray with holder
Gel comb(s)

PROTOCOL:

- Add agarose to TAE Buffer

- Microwave at full power for 1.5 minutes

- Swirl the solution briefly

- Microwave for another minute at full power

- Swirl briefly again

- Set up Gel tray in the holder

- Add GelRed to the tray

- Add ~50mL of agarose solution to the holder

- Store remain agarose solution at 59°C

- Mix agarose solution gently with a gel comb

- Place gel comb(s) in the appropriate slots in the gel tray

- Allow 20-30 minutes for the gel to set

- Use immediately

Agarose Gel Electrophoresis

MATERIALS:

1μL 6x loading dye per sample
5μL of each sample
TAE buffer
0.8% Agarose gel
3μL DNA ladder

EQUIPMENT:

Electrophoresis apparatus
UV Gel reader w/ protective glass plate
UV filtering safety glasses

PROTOCOL:

- Add 1μL 6x loading dye to sample, pipette to mix. Repeat for all samples

- Fill electrophoresis apparatus with TAE buffer, insert gel

- Load DNA ladder to a well made by each comb

- Load 5μL sample into remaining wells

- Run apparatus for 30-40 minutes at a constant 90V

- Transfer gel to UV gel reader, cover with clear plastic

- Put on UV filtering safety glasses

- Read gel

SDS Gel Electrophoresis

MATERIALS:

0.5-1mL Lysed cells (protein samples)
1.5 mL Eppendorf tubes
4x LDS loading buffer (volume equal to 1/3 total protein sample volume)
500μL 1x LDS loading buffer per sample
Bolt 4-12% Bis-Tris Plus gel(s), 15 well
~500mL SDS buffer
5μL 1kDa Protein ladder per gel
~75-100mL Coomassie Blue staining solution
~150-200mL Destaining solution

EQUIPMENT:

Centrifuge
Vortex machine
Thermocycler or heating block
Electrophoresis apparatus
False back plate (if running an odd number of gels)
Putty knife
Shaking table
Gel imager

PROTOCOL:

- Spin down lysed cells at 12 000g and 4°C for 10 minutes

- Pipette out as much of the supernatant as possible, into new tubes

- Add 4x LDS loading buffer to supernatants in a 1:3 ratio

- Add 500μL 1x LDS loading buffer to each of the pellet samples

- Invert supernatant samples 3-4 times to mix

- Vortex pellet samples until dissolved into LDS buffer

- Heat shock samples at 95°C for 5 minutes

- Centrifuge samples for 10 minutes at 12 000g and 4°C

- Set up electrophoresis apparatus, using false back plate if not running a second gel in the same apparatus

- Remove Bolt gel from packaging, remove tape and comb from gel

- Place Bolt gel into electrophoresis apparatus facing the centre, secure the gel and back plate (if using) with provided mechanism

- Ensure the middle chamber is water-tight by adding a small amount of buffer to the chamber and checking for leaks, re-secure and re-test chamber if leaks are detected

- Fill the inner chamber until the buffer completely covers the gel wells

- Fill the outer chamber until the level of the buffer is 1cm above the exposed gel

- Load 5μL of protein ladder into a well of each gel

- Load 5μL of soluble sample into different wells, load only fully liquid portion, not gel portion

- Load 3μL of insoluble sample into different wells

- Close apparatus and start electrophoresis at a constant 220V for 35 minutes

- Remove gel from apparatus and remove plastic casing from gel using a putty knife

- Cut off the thick section of gel at the bottom of the gel

- Place gel in enough Coomassie blue solution to completely cover it

- Place container with gel and Coomassie blue solution onto shaking table at 120rpm for 1-2 hours

- Carefully decant Coomassie blue solution, add destaining solution until gel is completely covered

- Leave on shaking table at 120rpm for 1-2 hours

- Replace destaining solution, leave on shaking table for 16 hours

- Decant destaining solution and image with gel imager

Bacterial Precultures

MATERIALS:

3mL LB media
3μL 100mg/mL ampicillin stock
1 15mL falcon tube
Agar plate or freezer stock containing colonies of interest
Colony picking tool
Tape

EQUIPMENT:

Gas flame or biological materials hood
Shaking incubator

PROTOCOL:

- Add ampicillin stock to LB media in falcon tube

- Pick a colony or scratch the freezer culture with the picking tool while near a gas flame or in a biological materials hood

- Inoculate media with bacteria by dipping scraping into media and mixing briefly

- Leave cap unscrewed, secure with tape

- Place into shaking incubator at 37°C and 250 rpm for 16 hours

- Use within 24 hours of protocol completion

Bacterial Freezer Cultures

MATERIALS:

10mL Bacterial preculture
150μL Glycerol
850μL LB media
-80°C safe storage tubes

EQUIPMENT:

Centrifuge
-80°C freezer

PROTOCOL:

- Centrifuge bacterial preculture at 4 000g and 4°C for 10 minutes

- Gently decant and pipette out all LB media

- Add glycerol to LB media, shake vigorously to mix

- Resuspend cell pellet in 1mL 15% glycerol LB media

- Transfer to -80°C safe storage tube

- Transfer to -80°C freezer

Inducer Test Cultures

MATERIALS:

5mL LB media per testing condition
5μL 100mg/mL Ampicillin stock testing condition
50μL Bacterial preculture testing condition
Aluminum foil
50mL falcon tubes or similar incubation containers
Tape
20% arabinose, filter sterilized (volume dependent on testing conditions)

EQUIPMENT:

Baffled Erlenmeyer flask
Spectrophotometer
Shaking incubator
Centrifuge

PROTOCOL:

- Add 5mL LB media per testing condition (plus 5mL for the control) to the baffled Erlenmeyer flask

- Add 5μL 100mg/mL ampicillin per testing condition (plus 5μL for the control) to the LB media

- Add 50μL bacterial preculture per testing condition (plus 50μL for the control) to LB+amp media

- Incubate at 37°C and 250rpm

- Check cell OD₆₀₀ at 1.5 hours after inoculation and every 15 minutes thereafter until OD₆₀₀ reaches ~0.4

- Transfer 5mL of LB+amp culture aliquots into 50mL falcon tubes or similar incubation containers, leaving lids unscrewed, securing with tape if necessary

- Separate cultures into different testing conditions and induce with arabinose

- Allow cells to incubate in the testing conditions

- Harvest cells by centrifugation at 4 000g and 4°C for 10 minutes

- Decant media and analyze immediately or store at -20°C

Making Chemically-competent cells

MATERIALS:

Agar plate of untransformed DH5α cells
Colony picking tool
50mL LB media
Aluminum foil
22.5mL Ice-cold 50mM CaCl2 solution
Ice
0.25mL Glycerol
1.5mL Eppendorf tubes

EQUIPMENT:

250mL baffled Erlenmeyer flask
Gas flame or biological materials hood
Shaking incubator
Spectrophotometer

PROTOCOL:

- Pick a colony from the agar plate and inoculate LB media in a 250mL baffled Erlenmeyer flask with the culture while in a biological materials hood or near a gas flame

- Cover the flask with aluminum foil

- Incubate culture at 37°C and shaking at 250rpm until the OD₆₀₀ is between 0.35-0.4

- Centrifuge the culture for 10 minutes at 4 000g and 37°C

- Decant supernatant

- Resuspend cells in 20mL of CaCl₂ solution

- Incubate for 20 minutes on ice

- Centrifuge the culture for 10 minutes at 4 000g and 37°C

- Decant supernatant

- Mix glycerol with 2.5mL of CaCl₂

- Resuspend cells in CaCl₂-glycerol solution

- Separate the cells into 100μL aliquots in the Eppendorf tubes

- Use competent cells immediately or store at -80°C

DH5α Cell Transformation

MATERIALS:

Ice
100μL aliquot of chemically-competent cells
1-10ng of DNA vector dissolved in 1-5μL MQ water
0.9mL S.O.C. Media
LA+0.1mg/mL ampicillin plate

EQUIPMENT:

42°C water bath
Shaking incubator
Non-shaking Incubator

PROTOCOL:

- Thaw cells on ice if using frozen cells

- Add DNA solution to cell aliquot, tap gently to mix

- Incubate for 15 minutes on ice

- Heat shock cells in water bath for 45 seconds

- Incubate on ice for 2 minutes

- Add S.O.C. Media to cells, tap gently to mix

- Incubate cells at 37°C and 225rpm for 1 hour

- Plate 100μL of cells onto agar plate

- Incubate plate at 37°C for 16 hours

Taq PCR

MATERIALS:

16.5μL MQ water for each reaction
2.5μL x10 ThermoPol buffer for each reaction
0.5μL each of 10mM Forward and 10mM Reverse primers for each reaction
0.5μL dNTPs (10mM each dNTP) for each reaction
0.125μL 5 000 U/mL Taq polymerase for each reaction
PCR tube for each reaction
1μL DNA template (variable concentration) for each reaction

EQUIPMENT:

Thermocycler

PROTOCOL:

- Mix a master mix of sufficient volume for all desired PCR runs. Each reaction consists of:

MQ water
ThermoPol buffer
Forward primer
Reverse primer
dNTPs
Taq polymerase

- Split master mix into 24μL aliquots in PCR tubes

- Add DNA template to each reaction

- Run the thermocycler with the following program:

95°C for 3 minutes
30 cycles of:
- 95°C for 30 seconds
- 50°C for 30 seconds
- 68°C for 2.5 minutes
68°C for 5 minutes
Hold at 12°C

Colony PCR & Grid Plates

MATERIALS:

LA+0.1mg/mL ampicillin plate
46.5μL MQ water per analyzed colony
PCR tube per analyzed colony
Colony pick tool per analyzed colony
LA plate containing colonies
2.5μL x10 ThermoPol buffer per analyzed colony
0.5μL each of Forward and Reverse primers (10mM) per analyzed colony
0.5μL dNTPs (10mM each dNTP) per analyzed colony
0.125μL 5 000 U/mL Taq polymerase per analyzed colony
1μL x6 DNA loading buffer per analyzed colony
TAE buffer
0.8% Agarose gel
3μL DNA ladder per gel comb

EQUIPMENT:

Marker
Gas flame or biological materials hood
Incubator
Thermocycler
Electrophoresis apparatus
UV gel reader

PROTOCOL:

- Use the marker to make a numbered grid on the LA+amp plate

- Add 30μL MQ water to a number of PCR tubes equal to the number of colonies to be tested

- Near a gas flame or in a biological materials hood:

Use a pick tool to remove a colony from the LA plate
Scratch a grid square with the pick tool
Dip and spin the pick tool in a water-containing PCR tube
Repeat for the desired number of colonies

- Place the grid plate into a 37°C incubator

- Boil the picked colonies at 102°C for 10 minutes then hold at 12°C

- Mix a master mix of sufficient volume for all desired PCR runs. Each reaction consists of:

16.5μL MQ water
2.5μL ThermoPol buffer
0.5μL Forward primer
0.5μL Reverse primer
0.5μL dNTPs
0.125μL Taq polymerase

- Split master mix into 24μL aliquots in PCR tubes

- Add 1μL of each the boiled colonies to separate master mix tubes

- Run the thermocycler with the following program:

95°C for 3 minutes
30 cycles of:
- 95ºC for 30 seconds
- 50ºC for 30 seconds
- 68ºC for 2.5 minutes
- 68ºC for 5 minutes
- Hold at 12ºC

- Add 1μL 6x loading dye to 5μL of PCR product, pipette to mix. Repeat for all samples

- Fill electrophoresis apparatus with TAE buffer, insert gel

- Load 3μL DNA ladder to a well made by each comb

- Load 5μL sample into remaining wells

- Run apparatus for 30-40 minutes at a constant 90V

- Transfer gel to UV gel reader, cover with clear plastic

- Read gel

DpnI Digestion

MATERIALS:

17μL Sample
2μL Cutsmart buffer x10
1μL 20 000 U/mL DpnI
PCR tube or Eppendorf tube

EQUIPMENT:

Thermocycler, heating block or water bath

PROTOCOL:

- To 17μL of sample, add 2μL x10 Cutsmart buffer and 1μL DpnI

- Mix gently by tapping

- Incubate at 37°C by chosen heating apparatus for 1 hour

Growth Curve Expression Test

MATERIALS:

50mL LB Media per testing condition plus 50mL
50μL 100mg/mL ampicillin stock per testing condition plus 50μL
500μL Bacterial precultures of cultures of interest per testing condition plus 500μL
500μL Bacterial preculture of appropriate negative control per testing condition plus 500μL
Aluminum foil
~5mL 20% arabinose, filter sterilized per testing condition plus 5mL

EQUIPMENT:

Baffled flasks
Shaking incubator
Spectrophotometer

PROTOCOL:

- Add LB media per testing condition (plus 50mL for the control) to a baffled flask

- Add 100mg/mL ampicillin per testing condition (plus 50μL for the control) to the baffled flask

- Add bacterial preculture per testing condition to LB+amp media and mix thoroughly

- Transfer 50mL of LB+amp culture aliquots into 250mL Erlenmeyer flasks, covering the opening with aluminum foil

- Incubate at 37°C and 250rpm

- Check cell OD₆₀₀ and take a 1mL sample every 30 minutes after inoculation until OD₆₀₀ reaches ~0.4 for each culture, store samples at -20°C

- Induce with arabinose to a final arabinose concentration of 2%, induce cultures as they reach OD₆₀₀~0.4, not all at once

- Allow cells to incubate at 37°C and 250rpm

- Check cell OD₆₀₀, and take a 1mL sample every 15 minutes until 4 hours after induction, store samples at -20°C

Expression Cultures

MATERIALS:

200mL LB media
200μL 100mg/mL ampicillin stock
2mL Preculture
Aluminum foil
22mL 20% Arabinose stock solution

EQUIPMENT:

0.5L baffled flask
Shaking incubator
Spectrophotometer
Centrifuge

PROTOCOL:

- Add 100mg/mL ampicillin stock to LB media in the 0.5mL baffled flask

- Add preculture to the baffled flask, cover the opening with aluminum foil

- Incubate at 37°C and 250rpm shaking

- Check cell OD₆₀₀ every 30 minutes after inoculation until OD₆₀₀ reaches ~0.4

- Induce with 20% arabinose

- Allow cells to incubate at 37°C and 250rpm for 4 hours

- Harvest cells by centrifugation at 4 000g and 46°C for 10 minutes

- Decant supernatant

Cell Lysis by Sonication

MATERIALS:

5-10mL Cell Samples
0.625mL 80mM PMSF stock
500mL 10mM Tris-HCl stock
500μL 10mg/mL Lysozyme stock
Ice

EQUIPMENT:

Centrifuge
Sonicator w/ microprobe

PROTOCOL:

- Centrifuge cells samples for 10 minutes at 4°C and 4 000g

- Mix a lysis buffer of:

0.625mL PMSF stock
500mL Tris-HCl stock
500μL Lysozyme stock

- Decant supernatant from the cell samples

- Resuspend each cell sample in 2mL lysis buffer, store samples on ice

- Store remaining lysis buffer at -20°C

- Sonicate each sample while maintaining them on ice at 39% intensity, for 2 minutes with a 10 seconds on, 20 seconds off pulse setting

- Analyze samples immediately or store at -20°C

Gibson Assembly

MATERIALS:

1.5g PEG 8000
3 ml 1M Tris-HCl, pH 8.0
150μl 2M MgCl2
0.24μm syringe filters
25μl 1M DTT
50μl 10mM dNTPs
50μl 50mM NAD+
1μl T5 exonuclease (NEB Cat. #M0363S – 10units/μl)
31.25μl Phusion High Fidelity DNA Polymerase (NEB Cat. #M0530S – 2 units/μl)
250μl Taq Ligase (NEB Cat. #M0208S – 40 units/μl)
437.75 μl MilliQ water
100ng of insert DNA
The number of linearized vector molecules equal to 1/3 the number of DNA inserts

EQUIPMENT:

Tube rotator
Thermocycler, heating block or heating bath

PROTOCOL:

- Make isothermal start mix by mixing:

1.5g PEG 8000
3mL 1 M Tris-HCl, pH 8.0
150μl 2M MgCl2

- Put on tube rotator until PEG is in solution

- Filter sterilize 25μl 1M DTT and 405μl isothermal start mix

- Make a 2X Gibson assembly master mix by mixing:

The filter sterilized 25μl 1M DTT
The filter sterilized 405μl isothermal start mix
50μl 50mM NAD+
1μl T5 exonuclease (NEB Cat. #M0363S – 10units/μl)
31.25μl Phusion High Fidelity DNA Polymerase (NEB Cat. #M0530S – 2 units/μl)
250μl Taq Ligase (NEB Cat. #M0208S – 40 units/μl)
437.75 μl MilliQ water

- Mix by pipetting gently

- Make 100μl aliquots

- Store isothermal mix and 2x Gibson assembly master mix at -20°C

- Make the cloning mix by mixing:

100ng of insert DNA
The number of linearized vector molecules equal to 1/3 the number of DNA inserts
2x Gibson cloning mix equal to the combined volume of DNA insert and linearized vector

- Incubate for 1 hour at 50°C

- Transform cells immediately or store cloning mix at -20°C

Solar Cell Assembly

MATERIALS:

127mg I₂
10mL Ethylene Glycol
83mg KI
1mL 0.5M Tryptophane solution
15mL Falcon tube
1mL Cell pellet expressing lycopene
5mg TiO2
6mL MQ water
Two 3cm x 2.5cm ITO glass plates
5mL Ethanol
1 Lint-free cloth
Tape

EQUIPMENT:

Shaking incubator
Centrifuge
Multimeter
Non-shaking incubator (Optional)
Graphite pencil
2 Small butterfly clips
Chemical fume hood

PROTOCOL:

- Dissolve I₂ in Ethylene Glycol

- Dissolve KI in iodide-glycol solution, set aside

- Add tryptophan solution into falcon tube

- Add cell pellet to tryptophan and mix gently to suspend

- Add TiO2

- Shake for 2 hours at 37°C

- Centrifuge for 10 minutes at 1000rpm

- Wash pellet by:

Adding 2mL MQ water, shaking to mix
Centrifuge for 3 200 rpm for 15 minutes
Decant water
Repeat the above 3 steps 2 additional times

- Clean ITO plates with ethanol and lint-free cloth, allow to air dry

- Find the conductive side of the plates with the multimeter by measuring resistance

- Tape down the glass plates, conductive side up

- Add paste from above steps to one ITO plate and smear evenly

- Allow paste to dry, gently heating will speed the process

- Use graphite pencil to cover the second plate in a layer of graphite/graphene

- Remove tape

- Sandwich the plates together, conductive sides facing inward and having overhangs where the tape was

- Attach butterfly clips to the sides of the ITO glass plates, leaving the area previously covered with tape exposed

- Gently add iodide solution into the cell by pipetting onto the exposed overhang while in a chemical fume hood

- Gently loosen clips to allow iodide solution to flow throughout the interior of the cell

Lycopene Extraction

MATERIALS:

50mL Cell culture(s)
Glass pipettes
1mL Acetone per cell culture
1mL Reverse osmosis water per cell culture
One 2mL Eppendorf tube per cell culture
96-Plate (for Plate reader)

EQUIPMENT:

Spectrophotometer
Centrifuge
Chemical fume hood
Vortex machine
Plate reader

PROTOCOL:

- Measure the OD₆₀₀ of the cell cultures

- Centrifuge the cell cultures at 4 700g and 4°C for 10 minutes

- Decant media

- In a chemical fume hood, add acetone to cell cultures, close the container holding the cell culture.

- Vortex for 5 minutes

- Centrifuge samples again at 4 700g and 4°C for 10 minutes

- Add water to 2mL Eppendorf tubes

- In a chemical fume hood, use a glass pipette to transfer acetone supernatant directly into the water

- Shake briefly to mix

- Transfer the extracts to wells on the plate reader plate

- Measure the extract’s OD₄₇₂

Thin Layer Chromatography (TLC)

MATERIALS:

Acetone (volume varies)
~35mL of each cell culture to be analyzed
0.9mL Benzene per cell culture
1 piece of thin layer cellulose paper

EQUIPMENT:

Glass chromatography chamber
Spectrophotometer
Centrifuge
Vortex machine

PROTOCOL:

- Fill the chromatography chamber with acetone (around 4 cm from the bottom), remember to have the lid on to avoid evaporation.

- Measure OD₆₀₀ of the cell cultures, ensure that the remaining steps use the same number of cells from each culture

- Centrifuge the cell cultures at 4 700g and 4°C for 5 minutes

- Decant supernatant

- Resuspend the pellet in 10% acetone dissolved in benzene, vortex to resuspend

- Centrifuge the cell cultures at 4 700g and 4°C for 2 minutes

- Pipette 100μL of benzene solution on to a thin layer cellulose paper and let dry

- Place the paper in the chamber, be sure that the dried cell extract is not in the acetone solution

- Allow the chamber to rest until the acetone is 2/3 up the paper

- Remove the paper from the chamber and allow it to dry