Protocols

Here you can find all the protocols used by the wet lab during the course of this project.

General Protocols

Here are basic protocols, which were used in mainly all of the methods including buffers that are needed in all the protocols and the primers utilised for the different PCR reactions and their annealing temperatures.


Objective

Amplification of the PCR template should be achieved. The PCR reaction was used to set up the bioreporter and to amplify the produced cDNA of the DARPins.


Estimated bench time

30 min


Estimated total time

2 h


Safety Requirements

Prepare the PCR in the laminar flow to protect the agents from potential contaminations with DNases or other DNA fragments. Additionally, wear gloves to protect the reaction from DNases. Lab coat and goggles for personal protection.


Equipment

  • SimpliAmp™ Thermal Cycler
  • Autoclaved PCR tubes and lids

Material

  • Phusion™ Plus PCR Master Mix by Thermo Scientific™
  • Autoclaved water
  • Reverse primer
  • Forward primer

Protocol

  1. Prepare Master Mix according to the following table and always add one more reaction to the prepared Master Mix to have a buffer when dividing the Master Mix to the individual tubes.

    2. Component Concentration Volume for 1 reaction in [µL]
    Autoclaved Water 18
    2x Phusion Master Mix 25
    Forward Primer 0.5 µmol/L 2.5
    Reverse Primer 0.5 µmol/L 2.5
    Total 48
  2. Mix Master Mix well by pipetting up-and-down.
  3. Divide 48 μL of the Master Mix to individual PCR tubes and add 2 μL of sample including a negative control i.e. sterile water.
  4. Run the following PCR programme

Step Temp [°C] Time [sec] Cycles
Initial denaturation 98 30 1
Denaturation 98 10
Annealing X* 10 35
Elongation 72 45
Final Elongation 72 300 1

*The annealing temperature was calculated for each primer pair individual.


Objective

Performed after agarose gel is run to extract the DNA product from the agarose gel.


Estimated bench time

1 h


Estimated total time

1 h


Safety Requirements

Wear UV protective goggles and attach UV shield to the gel doc for the marking of the gel, while the UV is turned on to protect yourself from the radiation. Wear gloves throughout the experiment to protect yourself from residual ethidium bromide and UV radiation during the gel cutting. Lab coat and goggles for personal protection. Additionally, the NT1 buffer contains Guanidinium thiocyanate, which is a highly reactive compound when combined with bleach.


Equipment

  • Gel documentation system
  • Centrifuge
  • 1.5 mL eppendorf tubes
  • Macherey-Nagel NucleoSpin Gel and PCR Clean-up, Mini kit for gel extraction and PCR clean up
  • Disposable scalpel
  • Heat block

Material

  • Provided by Macherey-Nagel NucleoSpin Gel and PCR Clean-up, Mini kit for gel extraction and PCR clean up
  • 96 - 100 % Ethanol
  • Vortex

Preparation prior to protocol

  1. Add indicated amount on bottle of 96 - 100 % ethanol to NT3 buffe

Protocol

  1. Add 200 μL of NT1 per 100 mg of 1 % agarose gel.
  2. Incubate sample for 10 minutes at 50 °C and vortex briefly every 2 minutes.
  3. Load 700 μL of sample in column.
  4. Centrifuge for 30 sec at 11,000 x g.
  5. Discard flow-through.
  6. Repeat steps 3 to 5, if there is sample left.
  7. Add 700 μL of NT3 buffer to the column.
  8. Centrifuge for 1 min at 11,000 x g.
  9. Discard flow-through.
  10. Centrifuge for 1 min at 11,00 x g.
  11. Discard flow-through.
  12. Put column in new 1.5 mL eppendorf tube.
  13. Add 30 μL of NE buffer on the membrane and incubate for 3 min at 37 °C.
  14. Centrifuge for 1 min at 11,000 x g.
  15. Store sample at - 20 °C.

Objective

Performed after agarose gel is run to extract the DNA product from the agarose gel.


Estimated bench time

1 h


Estimated total time

1 h


Safety Requirements

Wear UV protective goggles and attach UV shield to the gel doc for the marking of the gel, while the UV is turned on to protect yourself from the radiation. Wear gloves throughout the experiment to protect yourself from residual ethidium bromide and UV radiation during the gel cutting. Lab coat and goggles for personal protection. The binding buffer is harmful if swallowed and to aquatic life with long lasting effects. It causes severe skin burns and eye damage.


Equipment

  • Gel documentation system
  • Centrifuge
  • 1.5 mL eppendorf tubes
  • GeneJet Gel Extraction Kit by Thermo Scientific™
  • Disposable scalpel
  • Heat block
  • Vortex

Material

  • Provided by GeneJet Gel Extraction Kit by Thermo Scientific™
  • 96 - 100 % Ethanol
  • 3 M sodium acetate, pH 5.2

Protocol

  1. Use clean scalpel and cut as close to the band as possible
  2. Add 1:1 binding buffer to the gel volume (1 µL:1 mg)
  3. Incubate for 10 min at 50 - 60 °C until the gel is dissolved (invert now and then during incubation)
  4. Vortex mixture
  5. Mixture should have a yellow colour indicating an optimal pH, if the colour is orange or yellow add 10 µL of 3 M sodium acetate -> pH will become 5.2
  6. Transfer up to 800 µL of the solubilized gel solution to the GeneJET purification column
  7. If the volume exceeds 800 µL have several centrifuge steps of 30 - 60 sec at >12000 x g
  8. Centrifuge for 1 min at >12000 x g
  9. Discard the flow-through and place the column back into the same collection tub
  10. Add 700 µL of Wash Buffer (ethanol previously added to WB)
  11. Centrifuge for 1 min at >12000 x g
  12. Discard the flow-through and place the column back into the same collection tube
  13. Centrifuge the empty GeneJET purification column at >12000 x g for an additional 1 min to completely remove residual wash buffer
  14. Transfer the GeneJET purification column into a clean 1.5 mL microcentrifuge tube
  15. Add 50 μL of elution buffer or nuclease free water to the centre of the purification column and centrifuge for 1 min.
  16. Discard the purification column and Store purified DNA at - 20 °C

Objective

Performed to separate the DNA samples according to size on a 1% agarose gel.


Estimated bench time

15 min


Estimated total time

1 h


Safety Requirements

Wear thicker gloves like Neopren due to usage of Ethidium bromide, which is a carcinogen. Lab coat and goggles for personal protection.


Equipment

  • Gel tray from Bio-Rad
  • Gel comb from Bio-Rad
  • Gel caster from Bio-Rad

Material

  • Agarose, ultrapure, Invitrogen, Thermo Scientific™
  • MilliQ wate
  • Ethidium bromide, Amresco VWR chemicals

Protocol

  1. Agarose is weighed on a scale e.g. 1 g
  2. The weighed agarose is added to 1x TAE buffer in Erlenmeyer flask or bottle e.g. 100 mL
  3. The gel is heated in the microwave and cooled down to ~50 °C
  4. Bigger batches of agarose gel can be prepared stored in an incubator at 50 °C
  5. Place gel tray into the gel caster
  6. The gel is cast into the tray (approximately 50 mL) and 1-2 drops of ethidium bromide are added
  7. Mix ethidium bromide into the gel by mixing it with the gel comb and put the gel comb in the gel (pay attention to size of gel comb according to sample volume)
  8. Let gel to solidify for ~30 minutes

Objective

Performed to separate the DNA samples according to size on a 1% agarose gel.


Estimated bench time

2 minutes per sample


Estimated total time

2 minutes per sample


Safety Requirements

Wear lab coat and goggles for personal protection.


Equipment

  • None

Material

  • DNA Gel Loading Dye (6X) by Thermo Scientific™

Protocol

  1. 6x loading dye is added to sample in a 1:6 ratio meaning 1 volume of loading dye to 5 volumes of sample
  2. Mix sample and loading dye by pipetting up-and-down

Objective

Performed to separate the DNA samples according to size on a 1% agarose gel.


Estimated bench time

2 minutes per sample


Estimated total time

2 minutes per sample


Safety Requirements

Wear lab coat and goggles for personal protection from the ethidium bromide and to protect the DNA.


Equipment

  • Gel chamber
  • Power supply

Material

  • Solidified agarose gel
  • Prepared samples
  • GeneRuler 1 kb DNA Ladder, ready-to-use by Thermo Scientific™

Protocol

  1. Load ~ 6 μL of DNA ladder
  2. Load ~30 μL of the prepared sample
  3. Ensure that the running buffer is high enough
  4. Run the gel for approximately 30 - 45 minutes at 110 - 120 V

Objective

Performed to separate the DNA samples according to size on a 1% agarose gel and take an image of the gel.


Estimated bench time

10 min


Estimated total time

10 min


Safety Requirements

Wear thicker neoprene gloves, lab coat and goggles for personal protection from the ethidium bromide.


Equipment

  • Gel documentation system from Bio-Rad

Material

  • 70 % Ethanol

Protocol

  1. Check agarose gel, if the loading dye line has passed far enough
  2. Take out agarose gel from chamber and tilt to remove excess liquid from ge
  3. Carry gel tray and gel over on paper tissues to gel documentation system
  4. Select Bio-rad image lab on the computer
  5. Select on Nucleic Acids and click on Ethidium Bromide gel
  6. Select position gel and agree to FIlter 1
  7. Position the gel
  8. “Run protoco” and print and save the image


Objective

Measurement of DNA, RNA or protein concentration.


Estimated bench time

5 min per sample


Estimated total time

5 min per sample


Safety Requirements

Wear gloves, lab coat and goggles for personal protection and protection of the sample from RNases, DNases, proteases or contaminants.


Equipment

  • Nanodrop

Material

  • Sample
  • Blank

Protocol

  1. Choose programme for concentration measurement either protein, DNA or RNA
  2. For DNA choose dsDNA or ssDNA
  3. Clean with ddH2O
  4. Put blank i.e. eluent of sample and blank
  5. Wipe blank away
  6. Repeat blank
  7. Put 1 - 2 μL of the sample and measure
  8. Mark down values including concentrations and measurements at specific wavelengths to assess the purity

Objective

Calibration of pH metre for following pH measurements.


Estimated bench time

30 min


Estimated total time

30 min


Safety Requirements

Wear gloves, lab coat and goggles for personal protection from acids and bases that are required for the calibration.


Equipment

  • pH-metre

Material

  • HCl
  • NaOH
  • pasteur-pipette

Protocol

  1. Turn on pH metre
  2. Wash pH metre with ddH2O
  3. Put pH metre into pH 7 solution
  4. Press Cal
  5. Wait for the pH to adjust and press Read at the right pH
  6. Wash pH metre with ddH2O
  7. Put pH metre into pH 4 solution
  8. Press Cal
  9. Wait for the pH to adjust and press Read at the right pH
  10. Start pH measurement

Objective

Transformation of TOP10/α5 E. coli is required for plasmid amplification and protein expression.


Estimated bench time

1 h


Estimated total time

18 h


Safety Requirements

Wear gloves, lab coat and goggles for personal protection and ensuring that no contamination of outside spaces with the GMO occurs.


Equipment

  • Ice
  • Heat block
  • Centrifuge

Material

  • NEB TOP10 competent E. coli cells
  • LB-kanamycin plates

Preparation prior protocol:

  1. Incubate LB-kanamycin plates at 37 °C
  2. Ensure that the SOC medium is not frozen

Protocol

  1. Thaw competent cells on ice
  2. Pipet 100 µl of competent cells and all of the (around 2-10 µl) of ligation reaction product into an eppendorf tube, mix gently by pipetting up-and-down
  3. Incubate tubes on ice for 30 minutes
  4. Incubate tubes in 42°C for 30 sec in the heat block
  5. Incubate tubes on ice for 5 minutes immediately
  6. Add 700 µl of SOC medium
  7. Incubate cells at 37°C for 40 minutes
  8. Centrifuge for 3 mins at 3300 x g
  9. Remove about 450 µL supernatant
  10. Resuspend cells in the remaining supernatant and plate
  11. Incubate overnight at 37°C.

Objective

Preparation for buffers needed for different protocols indicated in the specific protocols.


Estimated bench time

20 min


Estimated total time

20 min


Safety Requirements

Wear gloves, lab coat and goggles for personal protection and for Magnesium Acetate, which can form combustible dust. EDTA can cause eye irritation. SDS is a flammable solid and combustible dust with acute inhalation toxicity targeting the respiratory system, forming dusts and mists, causing skin corrosion/ irritation, serious eye damage/ eye irritation. Imidazole is harmful if swallowed, causing severe skin burns and eye damage and may damage fertility and the unborn child.


Equipment

  • Schott bottle
  • Stir bar
  • pH metre
  • Beaker

Material

  • Specific for each buffer

Protocol

  1. Add some sterilised water into the beaker
  2. Add the ingredients indicated in the table in the right concentration
  3. Fill the beaker a bit below the volume needed for the buffer
  4. Adjust the pH, if necessary
Buffer Recipe Volume Amount pH
PBS 137 mM NaCl
2.68 mM KCl
8.1 mM Na2HPO4 x 2 H2O
1.47 mM KH2PO4		 2L 16 g NaCl
0.4 g KCl
2.88 g Na2HPO4 x 2 H2O
0.4 g KH2PO4 		7.4
TBS 50 mM Tris-HCl
150 mM NaCl (after pH adjustment) 		1L 6.1 g Tris-HCl
8.8 g NaCl 		7.4 at 4 °C
TBS-T TBS
0.05 % Tween-20 		400 mL 399.5 mL
0.2 mL 		N/A
WBT TBS-T
50 mM MgAc 		200 mL 190 mL TBS-T
10 mL MgAc 		7.5 at 4 °C
EB 50 mM Tris-Hcl
0.05 M EDTA 		200 mL 180 mL Tris-HCl
20 mL EDTA 		7.5 at 4 °C
Running Buffer (1x) 25 mM Tris-Base
192 mM Glycine
0.1 % SDS 		0.5 L 1.51 g Tris-Base
7.21 g Glycine
0.5 g SDS 		8.3
SDS 10% 10 % (m/v) in ddH2O 5 mL 0.5 g SDS N/A
APS 10% 10 % (m/v) in ddH2O 2 mL 0.2 g APS N/A
1x TAE 1x TAE 1L 20 mL 50x TAE N/A
Lysis buffer 20 mM Tris
500 mM NaCl
10 mM Imidazole 		500 mL 1.21 g Tris
14.61 g NaCl
0.34g Imidazole 		8
Buffer A 50 mM NaH_2PO_4 · 2H_2O
300 mM NaCl 		100 mL 0.78 g NaH_2PO_4 · 2H_2O
1.75 g NaCl 		8
Buffer B Buffer A 100 mL 0.78 g NaH_2PO_4 · 2H_2O
1.75 g NaCl
1.7 g Imidazole 		8
Wash Buffer 25 mM Imidazole
300 mM NaCl
50 mM NaH_2PO_4 · 2H_2O 		50 mL 45 mL Buffer A
5 mL Buffer B 		N/A
4x Separating gel buffer 1.5 M Trizma
1 M HCl
0.4 % SDS 		1L 182 g Trizma
350 mL 1 M HCl
4 g SDS 		8.8
4x Stacking gel buffer 0.5 M Trizma
1 M HCl
0.4 % SDS 		1L 61 g Trizma
400 mL 1 M HCl
4 g SDS 		6.8
Destaining solution 7.28 M Ethanol
1.75 M Acetic acid
Sterile water 		2L 850 mL Ethanol
200 mL Acetic acid
950 mL sterile water 		N/A
Coomassie Solution 1.2 mM SERVA blue R
7.28 M 99.9 % Ethanol
1.75 M Glacial acetic acid
Sterile water 		1L 1 g SERVA blue R
425 mL Ethanol
100 mL Glacial acetic acid
475 mL sterile water 		N/A filter-sterilise
EDTA 0.5 M EDTA
NaOH tablets 		250 mL 46.5 g EDTA
NaOH tablets 		8
4x SDS-PAGE sample buffer 200 mM Tris-HCl
40 % (v/v) Glycerol
8 % SDS
200 mM DTT 		10 mL 2 mL Tris-HCl
4 mL 99.5 % Glycerol
2 mL 20 % SDS
2 mL DTT 		6.8
5x SDS running buffer 0.025 M Trizma base
0.192 M glycine
1 % SDS 		2 L 30 g Trizma base
144 g glycine
10 g SDS 		8.3

Name Forward Primer Reverse Primer Annealing Temperature in [°C]
Bioreporter Part 1 5' C A T G C C A T G G C A C T C A T T T C T C C T C T T T T C A C T G T C 3' 5' C T A G T C T A G A C C A G G A T T T C G C C G C G T T G T C 3' 63
Bioreporter Part 2 5' C T A G T C T A G A A T G G G T A A T A C T G A A G C A T T A G C G 3' 5' A C A T G C A T G C T A T G A A T G C G G A T A A C C C A A C A C G 3' 60
Reverse Transcription 5' T A A T A C G A C T C A C T A T A G G G T C T A G 3' N/A (annealing temp not defined)
Sequencing overhangs 5' A C A C T C T T T C C C T A C A C G A C G C T C T T C C G A T C T T A A T A C G A C T C A C T A T A G G G T C T A G3' 5' G T G A C T G G A G T T C A G A C G T G T G C T C T T C C G A T C T T T A T T G C T C A G C G G T G G C A G 3' 65
GFP DARPin 5' G T G A C T G G A G T T C A G A C G T G T G C T C T T C C G A T C T T T A T T G C T C A G C G G T G G C A G 3' 5' C G G C T C G A G G G A T C C A A T G C G 3' 65

Ribosome Display Protocols

Here are the protocols which are needed for ribosome display to affinity select DARPins. The protocols are arranged according to the experiment order.


Objective

Production of mRNA-ribosome-peptide complex of DARPin for ribosome display.


Estimated bench time

10 min per DARPin


Estimated total time

4 h


Safety Requirements

Ensure that the working area is clean and no RNases are removed. Work in laminar flow recommended. Wear gloves, lab coat and goggles for personal protection and protection of the sample from RNases.


Equipment

  • Ice bucket
  • 37 °C water bath
  • Autoclaved filter tips

Material

  • PURExpress® kit from NEB
  • Nuclease-free water
  • anti-ssrA v1
  • RNase inhibitor by Thermo Scientific™

Protocol

  1. Thaw the necessary number of aliquots of solution A and B on ice
  2. Mix solutions well before usage especially solution A
  3. Pulse-spin in microfuge
  4. Assemble reaction according to the following table

    5. Ingredient Concentration Amount in [µL]
    Solution A 5
    Solution B (minus RF123) 3.75
    RNase inhibitor (protocol 20 U/µL) 0.3
    anti-ssrA v1 100 uM stock 0.75
    nuclease-free ddH2O fill up to 12.5
    DNA product 3 ng (protocol 250 - 1000 ng) 2.7
  5. Incubate at 37°C for at least 2 hours (max incubation time of 4 h may increase yield)
  6. Stop the reaction by placing the tube(s) on ice
  7. Use samples for analysis or purification or freeze at -20°C for use at a later time

Objective

Target immobilised in a 96-well plate for affinity selection of DARPins during the ribosome display.


Estimated bench time

1 h


Estimated total time

18 h


Safety Requirements

Wear gloves, lab coat and goggles for personal protection from EDTA, which can cause eye irritation.


Equipment

  • 96-well plate
  • Paper towels
  • Shaker

Material

  • TBS
  • TBST
  • TBS + 0.5 % BSA
  • WBT

Preparation prior protocol:

  1. Prepare TBS, TBST, TBS + 0.5 % BSA, WBT according to the buffer protocols

Protocol

  1. Wash the 96-well plate with TBS 3 times
    1. Add TBS volume ~300 µL and maybe swirl a bit
    2. Smash plate on clean paper towels
    3. Repeat
  2. Air-dry the plate
  3. Add 100 μL of 66 nM of a Avidin solution diluted in TBS
  4. Incubate overnight at 4 °C on a shaker in a humidified environment or for 1 h at 37 °C
  5. Wash the coated plate 3 times with TBS
  6. Add 300 μL of TBS containing 0.5 % BSA
  7. Incubate the plate for 1 h at room temperature on a shaker
  8. Discard solution from the plate and tap the plate upside-down on a clean paper towel
  9. Add 200 μL ~250 nM of biotinylated AIP diluted in TBS containing 0.5 % BSA
  10. Incubate the plate for 1 h at 4 °C
  11. Remove all remaining buffer
  12. Wash wells 3 times with 300 μL of TBST
  13. Wash the coated wells 3x with ice-cold WBT on ice
  14. Remove ice-cold WBT from the ice-cold microtiter plate shortly prior to the selection of binders

Adaptation of Protocol for un-biotinylated GFP

  1. Wash the 96-well plate with TBS 3 times
    1. Add TBS volume ~300 µL and maybe swirl a bit
    2. Smash plate on clean paper towels
    3. Repeat
  2. Air-dry the plate
  3. Add 100 μL of 66 nM of a un-biotinylated GFP solution diluted in TBS
  4. Incubate overnight at 4 °C on a shaker in a humidified environment or for 1 h at 37 °C
  5. Wash the coated plate 3 times with TBS
  6. Add 300 μL of TBS containing 0.5 % BSA
  7. Incubate the plate for 1 h at room temperature on a shaker
  8. Discard solution from the plate and tap the plate upside-down on a clean paper towel
  9. Wash wells 3 times with 300 μL of TBST
  10. Wash the coated wells 3x with ice-cold WBT on ice
  11. Remove ice-cold WBT from the ice-cold microtiter plate shortly prior to the selection of binders

Objective

Selection of binders is the affinity selection of DARPin to target during the ribosome display.


Estimated bench time

30 min


Estimated total time

1 h 30 min


Safety Requirements

Wear gloves, lab coat and goggles and ensure to always have new gloves to avoid RNases to not degrade the mRNA and to protect yourself from Tris-Acetate, which causes eye and skin irritation.


Equipment

  • Filter tips
  • Shaker

Material

  • Stopped translation reaction
  • WBT
  • 96-well plate with immobilised target

Preparation prior protocol:

  1. Prepare WBT according to the buffer protocols

Protocol

  1. Add aliquots of 150 μL of the stopped translation reaction
  2. Gently shake the microtiter plate for 1 h at 4 °C
  3. Beat the plate dry and wash 5 times with WBT

Objective

Elution of RNA from the mRNA-ribosome-protein-complex after the selection of binders during the ribosome display to isolate mRNA for sequencing and identifying, which DARPin bound the best.


Estimated bench time

5 min


Estimated total time

25 min


Safety Requirements

Wear gloves, lab coat and goggles to protect yourself from EDTA and Tris-Acetate in WBT, which both cause eye irritation and the latter also skin irritation.


Equipment

  • Filter tips
  • Shaker

Material

  • 50 μg/mL Saccharomyces cerevisiae RNA by Thermo Scientific™
  • Ice-cold EB

Preparation prior protocol:

  1. Prepare EB according to the buffer protocols

Protocol

  1. Add 100 µL of ice-cold Elution buffer containing 50 μg/mL S. cerevisiae RNA
  2. Incubate for 10 min on ice with gentle shaking
  3. Repeat the previous two steps

Objective

Purification of eluted RNA from ribosome display to prepare it for reverse transcription and sequencing.


Estimated bench time

1 h


Estimated total time

1 h


Safety Requirements

Prepare the RNA purification in the laminar flow used for RNase-free work to protect the agents from potential contaminations with RNases and DNases or additional DNA fragments. Additionally, wear gloves to protect the reaction from RNases and DNases.Lab coat and goggles for personal protection.


Equipment

  • Centrifuge
  • 1.5 mL eppendorf tube

Material

  • Thermo Scientific GeneJET RNA Purification Kit
  • 96 - 100 % Ethanol

Preparation prior protocol:

  1. Add instructed amount by kit of 96 - 100 % of ethanol to wash buffer 1
  2. Check Lysis Buffer for salt precipitation before each use. Re-dissolve any precipitate by warming the solution at 37°C, then cool back down to 25°C before use

Protocol

  1. Adjust the volume of the reaction mixture to 100 µL with nuclease-free water
  2. Add 300 µL of Lysis Buffer without β-mercaptoethanol or DTT
  3. Mix thoroughly by vortexing or pipetting
  4. Add 180 µL of ethanol (96-100%)
  5. Mix by pipetting
  6. Transfer the mixture to the GeneJET RNA Purification Column inserted in a collection tube
  7. Centrifuge the column for 1 min at ≥12000 x g
  8. Discard the collection tube containing the flow-through solution
  9. Place the GeneJET RNA Purification Column into a new 2 mL collection tube
  10. Add 700 µL of Wash Buffer 1
  11. Centrifuge for 1 min at ≥12000 x g
  12. Discard the flow-through
  13. Place the purification column back into the collection tube
  14. Add 600 µL of Wash Buffer 2
  15. Centrifuge for 1 min at ≥12000 x g
  16. Discard the flow-through
  17. Place the purification column back into the collection tube
  18. Add 250 µL of Wash Buffer 2
  19. Centrifuge for 2 min at ≥12000 x g
  20. Optional. If residual solution is seen in the purification column, empty the collection tube and re-spin the column for 1 min at maximum speed
  21. Discard the collection tube containing the flow-through solution and transfer the GeneJET RNA Purification Column to a sterile 1.5 mL RNase-free microcentrifuge tube
  22. Add 30 µL of nuclease-free water to the centre of the GeneJet column
  23. Centrifuge for 1 min at ≥12000 x g to elute RNA
  24. Immediately continue ribosome display protocol
  25. Next Step there: Denature at 70 °C for 10 minutes

Objective

Reverse transcribe mRNA to cDNA that can be amplified and sequenced to identify the DARPin with the highest affinity to AIP.


Estimated bench time

20 min


Estimated total time

2.5 h


Safety Requirements

Prepare the PCR in the laminar flow used for RNase-free work to protect the agents from potential contaminations with RNases and DNases. Additionally, wear gloves to protect the reaction from RNases and DNases. Lab coat and goggles for personal protection.


Equipment

  • SimpliAmp™ Thermal Cycler
  • Autoclaved PCR tubes and lids

Material

  • Reverse primer (see primer list at the bottom of the page)
  • High-Capacity cDNA Reverse Transcription Kit by Thermo Scientific™
  • RNase free water
  • Eluted mRNA
  • 25X dNTP Mix
  • MultiScribe™ Reverse transcriptase
  • RT Buffer
  • RNase inhibitor

Protocol

  1. Prepare master mix solution on ice and mix gently

    2. Ingredients for reverse mastermix 1X Concentration Volume for 1 reaction in [μL]
    Reverse primer 2.0
    25X dNTP Mix (100 mM) 0.8
    RNase inhibitor 1.0
    MultiScribe™ Reverse transcriptase 1.0
    RT Buffer 2.0
    RNase free water 3.2
    Total 10.0
  2. Pipet 10 µL of 2X RT master mix into each well of a 96-well reaction plate or individual tube.
  3. Pipet 10 µL of RNA sample into each well, pipette up and down two times to mix. The RNA amount can't go over 2μg per 2X reaction volume.
  4. Seal the plates or tubes.
  5. Briefly centrifuge the plate or tubes to spin down the contents and to eliminate any air bubbles.
  6. Place the plate or tubes on ice until you are ready to load the thermal cycler.
  7. Program the thermal cycler using the conditions below.

    8. Settings Step 1 Step 2 Step 3 Step 4
    Temp. 25°C 37°C 85°C 4°C
    Time 10 min 120 min 5 min Hold

Objective

Sequencing of DARPins after ribosome display identifies which DARPins had the highest affinity to AIP.


Estimated bench time

30 min


Estimated total time

2 h


Safety Requirements

Prepare the PCR in the laminar flowto protect the agents from potential contaminations with DNases or additional DNA fragments. Additionally, wear gloves to protect the reaction from DNases. Lab coat and goggles for personal protection.


Equipment

  • SimpliAmp™ Thermal Cycler
  • Autoclaved PCR tubes and lids

Material

  • Reverse primer + overhang
  • Forward primer + overhang
  • dNTP
  • Phusion DNA polymerase
  • Phusion HF buffer
  • DMSO
  • Sterile water
  • cDNA

Protocol

  1. Amplify reverse transcription products according to:

    2. Ingredients Concentration Amounts for 1 reaction per well in [μL]
    Forward Primer 10 μM 0.25
    Reverse primer 10 μM 0.25
    dNTP 5 mM each 2
    Phusion DNA polymerase 2 U/μL 0.5
    Phusion HF buffer 5 x 10
    DMSO 2.5
    Water 29.5
    Total 45
  2. Add 5 μL of cDNA to the Master Mix
  3. Perform a hot-start PCR according to thermocycling programme:

    4. Step Temp [°C] Time [sec] Cycles
    Initial denaturation 98 30 1
    Denaturation 98 10
    Annealing X* 45 35
    Elongation 72 45
    Final Elongation 72 120 1
  4. Reduce number of cycles by 5 every new selection round
  5. Verify quality of PCR product on a 1.5 % agarose gel

Bioreporter Protocols

Here are the protocols for the bioreporter that will express GFP upon sensing AIP.


Objective

Purification of engineered plasmid for further experiments.


Estimated bench time

1 hour


Estimated total time

1 hour


Safety Requirements

Wear gloves, lab coat and goggles for personal protection from lysis buffer that can cause eye and skin irritation and for the protection of the samples from DNases.


Equipment

  • Centrifuge
  • 1.5 mL eppendorf tubes

Material

  • GeneJET Plasmid Miniprep Kit by Thermo Scientific™

Preparation prior protocol:

  1. NB! Once the RNase A solution has been opened always keep it in 4 degrees after that. And when used KEEP ALWAYS ON ICE!
  2. Add the RNase A solution to the Resuspension solution
  3. For 50 preps: Add 35 mL ethanol (96-100%) with 20mL of Wash solution => total volume: 55mL
  4. Check that there is no salt precipitation in the Lysis solution and Neutralisation solution. In case there is any, warm it in the incubator 37 °C for a while until it has dissolved and let cool down to RT again. DO NOT SHAKE THESE VIGOROUSLY.

Protocol

  1. Take 5 mL of the E.coli LB culture and put it in a Falcon tube
  2. Centrifuge it for 6,800 x g for 2 min
  3. Discard the supernatant and remove as much of the liquid as possible
  4. Add 250 μL of Resuspension Solution
  5. Resuspend the cell pellet, by pipetting or vortexing. NO CLUMPS ARE ALLOWED!!
  6. Add 250 μL of Lysis solution
  7. Mix the tube by inverting it 6-8 times. DON'T VORTEX. Do this until the solution becomes viscous and slightly clear.
  8. Incubate at room temp for 3min, until it appears clear
  9. Add 350 μL of the Neutralisation solution
  10. Mix by inverting the tube 6-8 times, clumps will be forming, DON'T VORTEX
  11. Centrifuge for 5 min at 12,000 x g at room temp, repeat again if supernatant is not completely clear
  12. Place GeneJET Spin Column into a collection tube, and pipet the supernatant into the column
  13. Centrifuge for 1 min at 11,000 x g
  14. Discard flow through, place column back to collection tube
  15. Add 500 μL of Wash solution
  16. Centrifuge for 1 min at 11,000 x g
  17. Discard flowthrough and place the column back in the same collection tube
  18. Repeat step 15-17
  19. Centrifuge for 2 min at 11,000 x g and discard the collection tube
  20. Place the nucleospin column into a 1,5mL microcentrifuge tube
  21. Add 50 μL of Elution buffer to the spin column, do not touch the membrane
  22. Incubate in RT for 2-3 min
  23. Centrifuge 2 min at 11,000 x g
  24. Store the purified plasmid in -20 °C

Objective

Purification of engineered plasmid for further experiments.


Estimated bench time

1 hour


Estimated total time

1 hour


Safety Requirements

Wear gloves, lab coat and goggles for personal protection from lysis buffer that can cause eye and skin irritation and for the protection of the samples from DNases.


Equipment

  • Centrifuge
  • 1.5 mL eppendorf tubes

Material

  • NucleoSpin Plasmid, Mini kit for plasmid DNA by Macherey-Nagel

Preparation prior protocol:

  1. NB! Once the RNase A solution has been opened always keep it in 4 degrees after that. And when used KEEP ALWAYS ON ICE!
  2. Add the RNase A solution to the Resuspension solution
  3. For 250 preps: Add 100 mL ethanol (96-100%) to the A4 Wash solution
  4. Check that there is no salt precipitation in the Lysis solution and Neutralisation solution. In case there is any, warm it in the incubator 37 °C for a while until it has dissolved and let cool down to RT again. DO NOT SHAKE THESE VIGOROUSLY.

Protocol

  1. Take 5 mL of the E.coli LB culture and put it in a Falcon tube
  2. Centrifuge it for 30 sec 11,000g --> or 6000g for 1 min
  3. Discard the supernatant and remove as much of the liquid as possible
  4. Add 250microL of Buffer A1
  5. Resuspend the cell pellet, by pipetting or vortexing. NO CLUMPS ARE ALLOWED!!
  6. Add 250 μL of Lysis solution
  7. Mix the tube by inverting it 6-8 times. DON'T VORTEX. Do this until the solution becomes viscous and slightly clear.
  8. Incubate at room temp for 3min, until it appears clear
  9. Add 300microL of buffer A3.
  10. Mix by inverting the tube 6-8 times, until blue samples turn colourless completely. DON'T VORTEX
  11. Centrifuge for 5min at 11,000g at room temp, repeat again if supernatant is not completely clear
  12. Place NucleoSpin Column into a collection tube, and pipet 750microL of the supernatant into the column
  13. Centrifuge for 1 min at 11,000 x g
  14. Discard flow through
  15. Repeat step 13-15 until there is no remaining lysate
  16. Add 600microL of Buffer A4
  17. Centrifuge for 1 min at 11,000g - discard flow through and place the column back in the empty collection tube
  18. Centrifuge for 2min at 11,000g and discard the collection tube
  19. Place the nucleospin column into a 1,5mL microcentrifuge tube
  20. Add 50microL of Buffer AE.
  21. Incubate in 37 °C for 2-3 min
  22. Centrifuge 1 min at 11,000g

Objective

Digest plasmids or DNA fragments with restriction enzymes producing sticky ends to facilitate integration of the DNA fragment into the plasmid.


Estimated bench time

15 min


Estimated total time

95 min


Safety Requirements

Prepare the PCR in the laminar flow to protect the agents from potential contaminations with DNases. Additionally, wear gloves to protect the reaction from DNases. Lab coat and goggles for personal protection.


Equipment

  • Heat Block

Material

  • Nuclease-free water
  • Restriction enzymes
  • Buffer (adjusted to according restriction enzyme)
  • PCR product
  • Plasmid template

    • Restriction Enzyme Thermal Inactivation at 65 °C Thermal Inactivation at 80 °C
    Xbal X
    Pael X
    Xhol X
    PstI X
    NcoI X
    EcoRI X

Protocol

  1. Assemble reaction according to the following table:

    2. Component Stock Concentration Concentration Volume for 1 reaction in [µL]
    Nuclease-free water Fill up to a total volume of 20 μL
    Buffer 10x 1x 2
    Restriction enzyme 1 20 U/μL 2 U/μL 2
    Restriction enzyme 2 20 U/μL 2 U/μL 2
    DNA template 0.1 - 0.5 μg X*

    * DNA template volume is adjusted according to the concentration of the sample.

  2. Mix reaction well by pipetting up-and-down.
  3. Incubate reaction for 1 h at 37 °C.
  4. Heat inactivate reaction for 20 minutes at a temperature specific for the restriction enzyme; see the table below:

    5. Restriction Enzyme Buffer O Buffer R EcoRI buffer FastDigest® buffer Tango Buffer
    Xbal X
    Pael X X
    Xhol X
    PstI X
    NcoI X
    EcoRI X
  5. Instead of heat inactivation the digested product can be directly run on the gel.

Objective

Ligate previously digested plasmids or DNA fragments that present usually sticky ends (sometimes blunt ends) again.


Estimated bench time

15 min


Estimated total time

95 min


Safety Requirements

Prepare the restriction digest in the laminar flow to protect the agents from potential contaminations with DNases. Additionally, wear gloves to protect the reaction from DNases. Lab coat and goggles for personal protection.


Equipment

  • Heat Block

Material

  • Nuclease-free water
  • Rapid DNA ligation kit by Thermo Scientific™
  • Restricted PCR product
  • Restricted Plasmid

Protocol

  1. Add ligation reaction according to the table:

    2. Product Concentration Volume in [µL]
    Plasmid X* X**
    Insert X* X**
    Rapid Ligation Buffer 5x 4
    T4 DNA Ligase 5 U/µL 1
    Nuclease-free water Add up to 20

    * measure the DNA concentration to assess the volume it should be 20 - 100 ng/μL for the plasmid

    **calculate the volume in a 3:1 ratio with the NEB calculator

  2. Vortex and spin down briefly
  3. Incubate for 1 to 2 h at 22 °C
  4. Heat-inactivate the reaction by incubating it for 20 min at 65 °C

Objective

Measuring the intensity of GFP signal from expression cells.


Estimated bench time

2-3 h


Estimated total time

72 h


Safety Requirements

Wear gloves, lab coat and goggles for personal protection and ensuring that no contamination of outside spaces with the GMO occurs.


Equipment

  • BioTek Synergy H1 Plate Reader
  • Spectrophotometer
  • Shaking incubator
  • 96-well plate

Material

  • Transformed expression cells with target plasmid
  • Control cells without target plasmid
  • LB media
  • Kanamycin antibiotic
  • AIP peptide solution (or other induction solution)
  • Sterile water

Preparations prior protocol:

  1. Inoculate the expression cells with target plasmid into 4 ml of LB media with 4 ̣µL of kanamycin (or other appropriate antibiotic)
  2. Incubate cells in 37 degrees and 220rpm overnight

Protocol

  1. Add 80uL of the pre-culture into new 4ml of LB media with 4uL of kanamycin, and incubate at 37 degrees, 220rpm shaking until OD600 reaches 0.6
  2. Take 10uL of the cultivated cells into 200uL of new LB media and add to the well on the 96-well plate (= 4.8*10^6 cells starting in each well)
  3. Add 10uL of the control cells also into 200uL of new LB media and add to the well
  4. Add 200uL of LB and 1uL of kanamycin to the wells that are designated blank wells
  5. Add water to all wells that are not in use
  6. Select and assign all wells on the BioTek Synergy H1 Plate Reader
  7. Set temperature for the run at 37 degrees
  8. Select Shake orbital
  9. For growth, select measurement at OD600, and for GFP intensity select measuring at 470 and 511nm wavelength (excitation and emission wavelength for GFP). For fluorescence measurement select measurement from the bottom, to ensure good readings
  10. Measure every 20 min

Expression System

Here are protocols used to both express DARPins and GFP.


Objective

Extraction of proteins from an induced E. coli liquid culture.


Estimated bench time

20 min


Estimated total time

1.5 h


Safety Requirements

Wear gloves, lab coat and goggles for personal protection and protection from the imidazole contained in the lysis buffer, which is harmful if swallowed, causes severe skin burns and eye damage. May damage fertility and may damage the unborn child.


Equipment

  • Sonicator
  • Rotator

Material

  • Lysis buffer
  • Ice

Preparation prior protocol:

  1. Prepare lysis buffer according to the buffer protocols.

Protocol

  1. Lyse bacterial pellet in 10 mL of the lysis buffer
  2. Add suspension to 50 mL falcons and incubate for 20 minutes at RT in a rotator
  3. Sonicate on ice. Use six 10 second bursts at 200-300 W (16-20amp) with a 10 second cooling period between each burst. Use a sonicator equipped with a microtip
  4. Centrifuge for 30 minutes at 12 000 rpm at 4 °C
  5. Remove supernatant to a fresh tube and take a 5 μL sample and store at -20 °C for SDS
  6. Take sample from pellet and store at - 20 °C for SDS

Objective

Purification of expressed protein from bacterial culture.


Estimated bench time

1 h


Estimated total time

1 h


Safety Requirements

Wear gloves, lab coat and goggles for personal protection from imidazole, PBS buffer and HisPurTM Ni-NTA Resin. Imidazole is harmful if swallowed, causes severe skin burns and eye damage. May damage fertility and may damage the unborn child. PBS buffer can cause serious eye irritation and HisPurTM Ni-NTA Resin may cause allergy, asthma symptoms or breathing difficulties if inhaled, an allergic skin reaction, cancer, damage to the unborn child, damage to organs through prolonged or repeated exposure. Harmful to aquatic life with long lasting effects.


Equipment

  • Centrifuge
  • 2 mL eppendorf tubes
  • 1.5 mL eppendorf tubes

Material

  • Ni-NTA spin column (His-tag) purification from Qiagen
  • Lysed bacterial cells

Preparation prior protocol:

  1. Prepare buffer A and B according to the buffer protocols

Protocol

  1. One column can contain around 750 μL of the lysed solution
  2. Take 350 μL of sample supernatant and 350 μL of buffer A into an eppendorf tube and mix by inverting the tube few times. If you have a lot of sample you can take a bigger falcon tube and place 5ml of sample and 5ml of buffer A into the tube.
  3. Place the 700 μL of mixture into the Ni-NTA column
  4. Incubate in RT for 5 min
  5. Centrifuge the column for 1 min at 3600 x g
  6. Discard the flowthrough
  7. If you have more sample, repeat step 3 to 5 until all sample has been used
  8. Add 500 μL of wash buffer
  9. Centrifuge 1min 3600 x g
  10. Discard flowthrough
  11. Repeat step 8 - 11
  12. Remove the collection tube and replace it with a new 1.5 mL eppendorf tube
  13. Add 100 μL of buffer B
  14. Incubate for 5min in RT
  15. Centrifuge for 1 min 3600 x g
  16. Save and remove the eppendorf tube

Objective

Inducing the expression of a protein in a GMO.


Estimated bench time

1 h


Estimated total time

6 h


Safety Requirements

Wear gloves, lab coat and goggles for personal protection from GMOs.


Equipment

  • 2x 2 L Erlenmeyer flasks
  • Spectrophotometer
  • Incubator
  • Centrifuge

Material

  • LB-kanamycin media
  • LB media
  • 1 M IPTG

Preparation prior protocol:

  1. Add 50 μL of kanamycin to 50 mL of LB

Protocol

  1. Add few colonies to ~50 mL of LB-kanamycin media and incubate at 37 °C at 250 rpm overnight
  2. Add 1 % of the overnight culture into fresh LB media and incubate at 37 °C and 250 rpm
  3. Measure OD at 600 and shake at 37 °C and 250 rpm until OD 600 is 0.5 - 0.6
  4. Move flasks to 250 rpm shaker at RT for 15 minutes (not always performed)
  5. Add 100 µM of IPTG and incubate for another 4 h
  6. Collect media via centrifugation in centrifugation bottles at 4000 x g at 4 °C for 45 minutes
  7. Discard the supernatant and store pellet at 4 °C

Objective

Recharging of Ni-NTA spin columns to perform protein purification with the same spin columns again.


Estimated bench time

1 h


Estimated total time

16 h


Safety Requirements

Wear gloves, lab coat and goggles for personal protection from EDTA, which can cause eye irritation and NiSO4 is harmful if swallowed, inhaled, causes skin irritation and damage to organs through prolonged or repeated exposure, may cause allergy or asthma symptoms, breathing difficulties if inhaled, cancer, damage to the unborn child or fertility and is suspected to cause genetic defects.


Equipment

  • Ni-NTA spin columns
  • Centrifuge

Material

  • 0.1 M EDTA
  • Sterile water
  • NiSO4
  • Buffer A

Preparation prior protocol:

  1. Prepare buffer A according to the buffer protocols

Protocol

  1. Optional) Soak column in 0.1 M EDTA overnight
  2. Discard EDTA
  3. Add 400 μL of 0.1M EDTA
  4. Centrifuge for 1 min 3600 x g - discard flowthrough
  5. Add 400 μL of sterile water
  6. Centrifuge for 1 min 3600 x g - discard flowthrough
  7. Repeat step 5 - 6
  8. Add 400 μL of 0.1 M NiSO4
  9. Incubate for 30 min at RT
  10. Centrifuge for 1 min 3600 x g - discard flowthrough
  11. Add 400 μL of sterile water
  12. Centrifuge for 1 min 3600 x g - discard flowthrough
  13. Add 400 μL of Buffer A
  14. Centrifuge 1 min 3600 x g - discard flowthrough
  15. Soak the column in 0.1 M EDTA and store at 4 °C

Objective

For the preparation of an SDS-gel the assembly of the chamber is needed both for casting and running the SDS gel.


Estimated bench time

20 min


Estimated total time

20 min


Safety Requirements

Wear gloves, lab coat and goggles for personal protection from the ethanol while cleaning the gel chamber. The actual gel chamber set-up takes place on the bench top.


Equipment

  • SDS-gel chamber by Thermo Scientific™
  • whatman-paper

Material

  • 70 % ethanol
  • water

Protocol

  1. Clean 1 small and 1 large glass plate with 0.75 mm spacers with ethanol and leave to dry
  2. Assemble glass slides on tabletop of bench and insert into the chamber (important white part goes into the centre)
  3. Insert glas slides into the chamber turning the short glass to the inside
  4. Clip the side pieces to ensure that the glass-slides are securely fastened
  5. Check that the glass slides are levelled at the bottom
  6. Put the chamber on top of the white platform (careful the white handles on the side need to be pulled out when putting it on)
  7. Safely secure the gel chamber by turning the white handles at the side 90 - 180 degrees in opposite directions (you feel when it is safely adjusted)
  8. You can test with water that the set-up does not leak, but ensure that you invert the gel to empty out the water and dry residual water with whatman-paper
  9. Mark the border with a pen to separating and stacking gel by measuring 0.5 cm below the comb
  10. Take out the comb and transfer the gel chamber to the fume hood for casting

Objective

For the preparation of an SDS-gel to separate protein samples according to size.


Estimated bench time

30 min


Estimated total time

30 min


Safety Requirements

Work in the fume hood and wear gloves, lab coat and goggles for personal protection from acrylamide, which causes serious damage to health by prolonged exposure through inhalation, in contact with skin and if swallowed, is toxic in contact with skin and if swallowed and may cause heritable genetic damage and cancer. Acrylamide/Bis-acrylamide, which causes eye irritation, is harmful if swallowed, inhaled, or absorbed through skin, may cause allergic skin reaction, cancer, nervous system damage, may form explosive dust-air mixtures and is light and air sensitive. APS may intensify fire, is an oxidizer, is harmful to aquatic life with long lasting effects and if swallowed or in contact with skin, causes skin and serious eye irritation, may cause respiratory irritation, allergy or asthma symptoms or breathing difficulties if inhaled. TEMED highly flammable liquid and vapour and causes severe skin burns, eye damage, may cause respiratory irritation, drowsiness or dizziness and is harmful if swallowed or if inhaled. Isopropanol is highly flammable liquid and vapour, causes serious eye irritation and may cause drowsiness or dizziness.


Equipment

  • SDS-gel chamber by Thermo Scientific™
  • whatmam-paper

Material

  • 70 % ethano
  • Water
  • 4x separating gel buffer
  • 40 % acrylamide
  • 10 % APS
  • TEMED
  • Isopropanol

Preparation prior protocol

  1. Prepare the 4x separating gel buffer and 10 % APS according to the buffer protocols.

Protocol

  1. Mix the separating gel in a 50 mL falcon tube according to the table below (enough for 2 gels):
    acrylamide end concentration in [%] 4x separating gel buffer in [mL] ddH2O in [mL] 40 % acrylamide in [mL]
    12.5 3.75 6.56 4.69
    14 5.00 8.00 7.00
  2. APS and TEMED are responsible for the polymerization, so be fast after adding them
    acrylamide end concentration in [%] 10 % APS in [µL] TEMED in [µL]
    12.5 90 18.75
    14 80 20
  3. Mix the solution by either closing the tube and shaking it or using a pasteur pipette to pipette up-and-down (try to avoid bubbles forming)
  4. Pipette the mixture with a pipette or pasteur pipette between the glass slides (remember it is easier to pipette at an angle and turn the glass slides so that the lower one will show towards you when pipetting)
  5. Add 500 µL of isopropanol evenly on top of the gel
  6. Let the gel solidify (takes around 45 minutes)
  7. Mix the stacking gel in a 50 mL falcon tube according to the table below (enough for 2 gels):
    acrylamide end concentration in [%] 4x separating gel buffer in [mL] ddH2O in [mL] 40 % acrylamide in [mL]
    3 2.85 7.65 0.9
  8. Pour isopropanol out and rinse with ddH2O
  9. Dry with Whatman-paper
  10. APS and TEMED are responsible for the polymerization, so be fast after adding them
    acrylamide end concentration in [%] 10 % APS in [µL] TEMED in [µL]
    3 90 18.75
  11. Mix the solution by either closing the tube and shaking it or using a pasteur pipette to pipette up and down (try to avoid bubbles forming)
  12. Pipette the mixture with a pipette or pasteur pipette between the glass slides
  13. Let the gel solidify (takes around 20 minutes)
  14. You can use the gel for running right away or store it at +4 °C wrapped in moist paper and plastic foil

Objective

For the preparation of an SDS-gel to separate protein samples according to size.


Estimated bench time

30 min


Estimated total time

2 h


Safety Requirements

Wear gloves, lab coat and goggles for personal protection and due to working with SDS, which is a flammable solid and combustible dust with acute inhalation toxicity targeting the respiratory system, forming dusts and mists, causing skin corrosion/ irritation, serious eye damage/ eye irritation. DTT, which causes eye, skin, and respiratory tract irritation and is harmful if swallowed.


Equipment

  • Centrifuge/ Minifuge
  • Heat Block

Material

  • 3x sample buffer
  • Colour protein standard

Protocol

  1. Take the gel out of the refrigerator, unpack and leave it to warm up (15 - 30 min)
  2. Prepare the samples (12 µL) for the SDS PAGE with the 3x sample buffer (6 µL)
  3. Incubate the tubes with the samples (NOT the protein standard) at 95°C for 5 minutes
  4. Spin down briefly at maximum speed

Objective

Separating proteins according to their molecular mass to confirm successful protein expression.


Estimated bench time

30 min


Estimated total time

2 h


Safety Requirements

Wear thicker neoprene gloves, lab coat and goggles for personal protection and for protection of agents in the prepared agarose gel. Refer back to “Casting the gel”


Equipment

  • Power supply
  • SDS-gel chamber by Thermo Scientific™

Material

  • SDS gel
  • 5x running buffer

Preparation prior protocol

  1. Prepare the 5x running buffer according to the buffer protocols.

Protocol

  1. Set up the running device with two gels on each side.
  2. Fill the inner chamber with 5x running buffer until it covers the glass plates. Avoid air bubbles. If any leaking occurs, reassemble the unit again.
  3. Fill the lower buffer chamber with 5x running buffer until the sandwiches are fully immersed.
  4. Carefully remove the comb, if air bubbles or residual acrylamide in the pockets remain, wash the pockets with 5x running buffer twice at the sink.
  5. Load samples.
  6. Run gel first at 80 V until the front reaches the separating gel border and the molecular weight standard has started to separate.
  7. Then run at 120 V until the front comes out in the bottom of the gel. Running the gel takes from 60 to 90 minutes.

Objective

Separating proteins according to their molecular mass to confirm successful protein expression and to visualise the run gel.


Estimated bench time

20 min


Estimated total time

20 min


Safety Requirements

Wear thicker neoprene gloves, lab coat and goggles for personal protection and for protection of agents in the prepared agarose gel. Refer back to “Casting the gel”


Equipment

  • Plastic box

Material

  • Distilled water
  • Destaining solution
  • Coomassie Brilliant solution

Preparation prior protocol

  1. Prepare the destaining solution and coomassie blue according to the buffer protocols.

Protocol

  1. Take the gel sandwich out of the electrophoresis chamber.
  2. Separate the glass plates from the gel
  3. Remove the stacking gel
  4. Loosen the gel from the plate by rinsing it with water and let the gel slip into the plastic box.
  5. Remove the water
  6. Add 20 mL of Coomassie Brilliant blue and incubate for 15 minutes (to a few hours) while gently shaking
  7. Remove excess rinsing it with small amount of ddH2O
  8. Destain with a destaining solution with gentle shaking overnight
  9. Place the gel into the distilled water to remove residual background stains
  10. Scan the gel


Objective

Freeze-drying a protein for transport. This prevents degradation of the protein at room temperature.


Estimated bench time

15 min


Estimated total time

2 days


Safety Requirements

Wear gloves, lab coat and goggles when working with liquid nitrogen. Extremely cold liquid (-196 °C) can cause severe frostbite and cold burns. Nitrogen gas can act as an asphyxiant as it dilutes the concentration of oxygen in air below the levels necessary to support life. Rescue workers may require self-contained breathing apparatus and protective clothing. Inhalation of nitrogen in excessive concentrations can result in dizziness, nausea, vomiting, loss of consciousness, rapid breathing, asphyxiation without warning and death and may cause severe cold burns and frostbite.


Equipment

  • Cryogenic container
  • Lyophilizer
  • 1.5 mL tubes with small holes in lid
  • Tongs
  • Lyophilizer jar

Material

  • Liquid Nitrogen

Protocol

  1. Put samples in liquid nitrogen in cryogenic container with lid
  2. Wait for less gas forming and take samples out with tongs
  3. Put lid with small holes on top of the frozen tubes
  4. Put samples in lyophilizer jar and attach to the lyoholizer
  5. Remove sample, if the bottom of the jar has reached room tempera