Difference between revisions of "Team:Manchester/InterLab"
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| − | <a href="https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf">CLICK TO VIEW</a> | + | <a style="color: purple;size: 20px;" href="https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf">CLICK TO VIEW</a> |
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Revision as of 00:15, 17 September 2018
InterLab Study
- The Fifth InterLab Study -
Measuring the GFP expression is a useful tool worldwide for quantifying gene expression. Currently, there are various differences in fluorescence data which is measured on different devices (e.g. different units of fluorescence recorded or different settings of the data processing used). The aim of the Fifth InterLab study is to make fluorescence measurements more compatible among various laboratories around the world. This is to be done by standardising the protocol for the machine calibration with Silica beads and relating OD600 values with colony forming units (CFUs). The expression of the GFP by one cell will be calculated by dividing the fluorescence of a population of cells via CFUs instead of using an OD600 measurement which is less precise.
- Protocols -
All InterLab protocols were provided by iGEM
CLICK TO VIEW
Materials (consumables):
LB broth (Luria Bertrani medium = rich media to grow bacteria)
TSS buffer (to prepare chemically competent cells)
S.O.C. medium (helps obtain the maximal transformation efficiency)
LB agar (gel where bacteria can grow)
Chloramphenicol (CAL) at stock concentration 25mg/ml
Preparation of chemical competent cells:
- Inoculate DH5α cells into 50mL LB and incubate at 37°C
- Monitor growth every 30 min by measuring optical density at 600nm (OD600) until it reaches OD600 = 0.4-0.6
- Harvest cells and prepare using 'TSS competent E.coli protocol' shown below:
LB Agar plates preparation:
- Prepare LB containing chloramphenicol (CAL) (at 25μg/ml)
- Melt LB in microwave (defrost setting for 15 min)
- Cool LB by running cold water over
- Stock of 25mg/ml CAL → so add 400μl CAL to 400ml LB
- Pour plates (in fume hood) and allow to solidify
Chemical Transformation:
- Add 1μl of DNA to 50μl of competent cells, mix well and place on ice for at least 30 min
- Heat shock cells at 42°C for 30 seconds, followed by 2 min incubation on ice
- Add 450μl of SOC medium to the cells and incubate for 45 min at 37°C (to allow protein expression (particularly the antibiotic resistance gene)
- Plate and spread (glass spreader sterilised over a flame and in ethanol) 50, 100, and 200μl of the cells into the agar plates made previously
- Incubate at 37°C for 2 hours
Materials (consumables):
Dimethyl sulfoxide (DMSO)
Polyethylene glycol (PEG)
MgCl2 stock solution
LB or SOC liquid medium
Ice
Methods:
- Inoculate DH5α E. coli cells into 50ml LB broth and incubate at 37°C
- Prepare the TSS buffer while waiting for the culture to grow and place it on ice (see: Table below)
- Monitor growth of the culture every 30 min by measuring the optical density at 600nm wavelength (OD600) until it reaches OD600 = 0.4-0.6 (takes approximately 2-3 hours)
- Once the proper optical density has been achieved, take 100ml of culture and centrifuge under 2.700xg for 10 min at 4°C
- Resuspend each tube in 5ml of pre-chilled TSS buffer with gentle vortexing
- Chill TSS suspended cells on ice for 15 min
- Aliquot 200μL of TSS suspended cells while ensuring the cells remain well mixed
- Cells can be used immediately or stored at -80°C
TSS Buffer Composition
| Component | Stock (M) | Amount |
|---|---|---|
| MgCl2 | 2 | 0.300ml |
| DMSO | - | 1ml |
| PEG (3350 or 8000) | - | 2g |
| LB Medium | to final volume of 20ml | to final volume of 20ml |
Full details found on: benchling
Materials (consumables):
2x Q5 Master Mix
10 µM forward primer
10 µM reverse primer
DNA template
Nuclease-free water
Methods:
- Gently mix the reaction from the components listed in the table below and place on ice
Note: addition of reagents are done in the following order to prevent degradation of primers: nuclease-free water, both primers, DNA, and then Q5 mix - When necessary, collect all liquid to the bottom of the PCR tube by spinning for a short time
- Transfer the PCR tube from ice to a PCR machine and begin thermocycling
PCR Reaction Components
| Component | 25µl Reaction | 50µl Reaction | Final concentration |
|---|---|---|---|
| Q5 High-Fidelity 2X Master Mix | 12.5µl | 25µl | 1X |
| 10µM Forward Primer | 1.25µl | 2.5µl | 0.5µM |
| 10µM Reverse Primer | 1.25µl | 2.5µl | 0.5µM |
| Template DNA | variable | variable | < 1,000ng |
| Nuclease-Free Water | to 25µl | to 50µl |
Thermocycling
The PCR machine should be set to run the following steps:
| Step | Temperature (°C) | Time |
|---|---|---|
| Initial denaturation | 98 | 30 seconds |
| 30 cycles | 98 (denaturation) 63 (annealing) see Note 1 72 (extension) |
5 seconds 30 seconds 27 seconds per kb |
| Final extension | 72 | 2 minutes |
| Hold | 10 | - |
Note 1: The NEB Tm calculator should be used to determine the annealing temperature when using the Q5 Master Mix: http://tmcalculator.neb.com/#!/
Full detail found at: NEB
Materials (consumables):
1% TAE Buffer
Agarose powder
SYBR Safe
Loading dye
DNA ladder
Make 1% agarose gel:
- Prepare 1% TAE agarose gel: dissolve 1g of agarose into 100ml of TAE buffer in a conical flask
- Warm in microwave for 1 min at max power
- Remove flask from microwave with care, swirl gently and cool under running tap
- Add 5µl of SYBR Safe
- Prepare a casting tray with suitable comb
- Pour to cool mixture into the casting tray and wait 15 min until it solidifies
Run gel:
- Add 5µl of PCR solution and 1µl 10x loading dye
- Load 6µl of DNA ladder alongside and all samples (Do not forget to add dye to ladder too) - NEB 1kb ladder used
- Run gel at 100V for 45 min
- Visualise gel on a transilluminator (SYBR Safe binds DNA and fluoresces under UV light)
Materials (consumables):
Restriction Enzyme: NEB enzyme finder to determine the restriction enzymes
10X Buffer: NEB double digest finder to determine the buffers that are required
Plasmid DNA
Nuclease-free water
Methods:
- Incubate the digestion reaction (see components below) for 1 hour at 37°C, then at 65°C for 20 min (to heat-inactivate the enzymes)
Restriction Digestion Mix
| Component | 50µl Reaction | Final concentration |
|---|---|---|
| 10X Buffer | 50µl | 1X |
| DNA | will vary | 250ng |
| Restriction Enzymes | 1µl (of each enzyme) | 10U |
| Nuclease-free water | to 50µl |
Materials (consumables):
T4 Ligase Buffer
T4 Ligase
Vector DNA
Insert DNA
Nuclease-free water
Ice
Methods:
- Allow the buffer to defrost on ice
- Calculate molar ratio for vector and insert DNA using NEBioCalculator (NEBioCalculator) (ideal ratio for insert:vector is 3:1)
- Make up ligation reaction as below
- If using T4 ligase: incubate at room temp for 1 hour
- NB: different ligases (eg. Quick Ligase) will require different incubation times
Ligation Reaction Composition
| Component | 10µl Reaction | Final concentration |
|---|---|---|
| 10X T4 Ligase Buffer | 1µl | 1X |
| Vecor DNA | vary | |
| Insert DNA | vary | |
| Nuclease-free water | to 10µl |
Materials (consumables):
Ethanol (96-100%)
Buffer PE
Buffer PB
Buffer EB
pH indicator I
Sodium acetate
QIAquick column
Loading dye
Notes before starting:
- Add ethanol (96-100%) to buffer PE before use (see bottle label for volume)
- All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional table-top microcentrifuge at room temperature
- Add 1:250 volume pH indicator I to Buffer PB. The yellow color of Buffer PB with pH indicator I indicates a pH of ≤7.5. If the purified PCR product is to be used in sensitive microarray applications, it may be beneficial to use Buffer PB without the addition of pH indicator I. Do not add pH indicator I to buffer aliquots
Methods:
- Add 5 volumes Buffer PB to 1 volume of the PCR reaction and mix. If the color of the mixture is orange or violet, add 10μl 3M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow
- Place a QIAquick column in a provided 2 ml collection tube
- To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 seconds until all the samples have passed through the column. Discard flow-through and place the QIAquick column back in the same tube
- To wash, add 0.75ml Buffer PE to the QIAquick column and centrifuge for 30–60 seconds. Discard flow-through and place the QIAquick column back in the same tube
- Centrifuge the QIAquick column once more in the provided 2ml collection tube for 1 min to remove residual wash buffer
- Place each QIAquick column in a clean 1.5ml microcentrifuge tube
- To elute DNA, add 50μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30μl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge
- 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
Materials (consumables):
Overnight culture
Buffer P1
Buffer P2
Buffer N3
Buffer PE
Sterile dH2O (MiliQ) water
Methods:
- Spin down the overnight cultures at 10000rpm for 10 min. Discard supernatant into virkon
- Resuspend the pelleted bacteria with 250μl of Buffer P1 (stored in fridge), and transfer the resuspended bacteria into a fresh 2ml eppendorf
- Add 250μl of Buffer P2 to the 2mL Eppendorf with bacteria and mix gently. Sample should turn blue (indicates cells have lysed)
- Incubate for 5 min at room temperature (do not exceed 5 min or plasmid will begin to degrade)
- Add 350μl of Buffer N3 and mix gently. Sample should be colourless and contain a white precipitant
- Centrifuge samples at 14,000rpm for 10 min using a table top centrifuge
- Transfer 750μl of the supernatant to a column placed on a 1.5ml Eppendorf tube (discard white precipitate). Centrifuge at 11,000rpm for 1 min using a table top centrifuge
- Discard the flow-through. Place column onto new eppendorf tube and add 750μl PE buffer (with added ethanol to the stock buffer if not already done so). Incubate at room temperature for 5 min. Centrifuge at 13,000rpm for 30 seconds
- Transfer column to fresh eppendorf. Centrifuge at 13,000rpm for 2 min (dry out)
- Transfer column into fresh eppendorf. Add 30μl of sterile dH20 (MiliQ) (add directly onto column to ensure water pushes DNA through) and incubate for 5 min at room temperature
- Centrifuge at 11,000rpm for 1 min. Do not discard this flow-through. This contains the extracted plasmid
Materials (consumables):
Buffer QG
Buffer PE
Buffer PB
3M Sodium Acetate
Sterile dH2O (MiliQ) water
Isopropanol
Methods:
- Excise the DNA fragment from the agarose gel with a clean, sharp scalpel
- Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100mg gel ~ 100μl). The maximum amount of gel per spin column is 400mg. For >2% agarose gels, add 6 volumes Buffer QG
- Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the 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
- Add 1 gel volume isopropanol to the sample and mix
- Transfer 750µl of supernatant to a column placed on a 1.5ml eppendorf. Centrifuge at 11,000 rpm for 1 min
- Discard flow through. Place column in a new Eppendorf tube. Add 500µl of PB buffer and centrifuge column at 13,000 rpm for 30 seconds
- Discard flow through. Place column in a new eppendorf. Add 750µl of PE buffer (check ethanol has been added, see: PCR Purification). Incubate at room temp for 5 mins, then centrifuge at 13,000 rpm for 30 seconds
- Transfer column into fresh Eppendorf. Centrifuge at 13,000 rpm for 2 mins
- Transfer column to new eppendorf. Add 30µl of MiliQ water and incubate for 5 mins at room temperature
- Final centrifuge at 11,000 rpm for 1 minute
- Test sample using Nanodrop (see below)
Nanodrop: calculate DNA concentration in sample
- Load 1μL of MiliQ water to the Nanodrop and blank. Clean and load another 1μl of MiliQ water, measure and proceed only if clean (absorbance is zero at all measured wavelengths)
- Load 1μl of sample and measure DNA concentration
- Check A260/280 ~ 1.8 and A260/230 ~2.0 (pure DNA sample should have values close to these numbers)
Materials (consumables):
LB liquid medium
Chloramphenicol at stock concentration of 25mg/ml
IPTG at stock concentration of 100mM
Tetracycline at stock concentration of 0.214mM
Arabinose at stock concentration of 100nM
Method:
Aseptic technique maintained at all times
- Transfer 50mL of LB +250L of chloramphenicol into 8x 250mL conical flasks.
- Inoculate 2 flasks labelled + and - with 200L of pSBC13eutS overnight culture.
- Grow the cultures at 37C in shaking incubator until and OD value of 0.5 is reached.
- Add IPTG, Tetracycline and Arabinose to the respective constructs in the amounts shown below.
- Incubate samples for 4hours in a 20C shaking incubator, remove 25mL of the culture and transfer to a falcon tube.
- Centrifuge at 9000rpm for 8minutes.
- Pour off supernatant, sample can be used immediately or frozen at -20C.
- Incubate samples for a further 16 hours at 20C (20 hours in total), transfer culture to a falcon tube.
- Centrifuge at 9000rpm for 8minutes.
- Pour off supernatant, sample can be used immediately or frozen at -20C.
Inducers to be added to Eut constructs
| Flask | Construct | Inducer and concentration to be added | |
|---|---|---|---|
| Flask 1 | pSBC13eutS- | None | |
| Flask 2 | pSBC13eutS+ | 250µM IPTG | |
| Flask 3 | pSBC13eutMN- | None | |
| Flask 4 | pSBC13eutMN+ | 1000 nM tetracycline | |
| Flask 5 | pSBC13eutSMN- | None | |
| Flask 6 | pSBC13eutSMN+ | 250µM IPTG and 100nM tetracycline | |
| Flask 7 | pSBC13eutLK- | None | |
| Flask 8 | pSBC13eutLK+ | 500µL arabinose |
Materials (consumables):
Primary and Secondary antibodies of choice
Nitrocellulose membrane (or other membrane of choice - note different membranes are suited to different sized proteins, but may follow slightly modified protocols to the one below)
Filter paper
iBind western system (ThermoFisher)
Method:
- Run samples on an SDS PAGE of choice (recommend at 200V for 45mins)
- Transfer protein from gel to membrane at 30V for 60-90mins. Assemble as shown in diagram below:
- Immerse blotted membrane in 5 mL 1X iBind™/iBind™ FD Solution.
- Dilute antibodies right after wetting the membrane
- Place the iBind™ Card on the stage and pipette 5 mL of 1X iBind™/iBind™ FD Solution evenly across the Flow Region.
- Add 1 mL of 1X iBind™/iBind™ FD Solution so that it pools at the center of the membrane region on the iBind™ Card.
- Place the membrane on top of the pooled solution with the protein-side down, and the low molecular weight region closest to the stack.
- Use the Blotting Roller to remove any air bubbles.
- Close the lid and add solutions sequentially to the iBind™ Wells starting with Well 1.
- Place the window cover over the viewing window, and close the well cover. Incubate 2.5 h to overnight.
- Rinse the membrane in water and proceed to immunodetection protocol.
Materials (consumables):
Phosphate Buffered Saline (PBS)
PBS + 2% paraformaldehyde
Method:
- Using a fresh transformation plate prepare overnights as follows:
10ml LB
10μl of each antibiotic as required (Chloramphenicol for localization tags and Ampicillin for Eut)
Inoculate media with a single colony
Grow overnight at 37⁰C - Prepare fresh liquid cultures as follows:
10ml LB
10μl of each antibiotic as required
Inoculate with 200μl of overnight culture - Grow to OD600 of 0.1, or for roughly 1 hour and induce the whole liquid culture with IPTG, Tet and/or Arabinose as required (IPTG for EutS, Tet for MN & Ara for LK. Tags are constitutive and don't need inducing).
Add 10μl of 100mM IPTG for 100μM, 2.34μl of 214μM Tet for 50nM & 10μl of 100mM Ara for 100μM. - Incubate induced samples at 30⁰C and take out at desired time-points (I found 2 and 3 hours are sufficient).
- At each time-point remove 1ml of each liquid culture and place in 1.5ml eppendorfs.
Spin down at 5000rpm for 2 minutes in a table top centrifuge and discard supernatant. - Resuspend pellet in 1ml PBS and spin down again at 5000rpm for 2 minutes, discarding supernatant.
- Next resuspend pellet in PBS+2% paraformaldehyde and leave at room temperature for 30 minutes.
- After 30 minutes, spin down at 5000rpm for 2 minutes and discard supernatant and resuspend pellet in 100μl of PBS.
-This suspension is now ready to make slides with.
Materials (consumables):
Fixed cell suspension
Blank microscope slides
Cover slips
Method:
- Pipette 12μl of suspension to a slide and add a cover slip.
- Press cover slip down slightly (this helps put all cells in the same plane so it's easier to focus the microscope).
- Allow to dry out of direct light to avoid photo bleaching (under blue roll is sufficient).
- Store somewhere dark like a slide box or wrapped in lens cloth.
-These slides are now ready to be visualized with fluorescence microscopy.
Analysis Modules:
ApplyThreshold
IdentifyPrimaryObjects
IdentifyPrimaryObjects
MeasureObjectSizeShape
ExportToSpreadsheet
Method:
-
-(This protocol is meant to inform CellProfiler users of our method of image analysis, not teach users how to use the software. For tutorials on how to use CellProfiler, visit: http://cellprofiler.org/examples/ or http://cellprofiler.org/tutorials/)
- Select the ‘Images’ option in the ‘Input modules’ window and drag and drop the target image into the file list.
- Select ‘NamesAndTypes’ in the ‘Input modules’ window and enter a value in the ‘Name to assign these images’ box, e.g. “raw_data” and click update.
- Right click in the ‘Analysis modules’ window and navigate to Add>All>ApplyThreshold. This will filter out any background noise. From the ‘Select the input image’ drop-down menu, select “raw_data” and name the output image, e.g. “ThreshGreen”. The threshold will need to be tweaked to match the picture, so set the ‘Threshold strategy’ to Manual and change the ‘Manual threshold’ value to match your image (the results can be previewed by clicking the ‘Start Test Mode’ button in the bottom left and using the ‘Step’ button to step through Analysis modules).
- Add another module by navigating Add>All>IdentifyPrimaryObjects. Select input as your threshold image e.g. ThreshGreen and name the objects to be identified e.g. Cells. Tweak the typical diameter range of the objects to roughly match your target objects. Select ‘Yes’ to discard objects outside the diameter range and select ‘No’ to discard objects touching the border. Like before set the threshold strategy to manual and tweak to match your image (Use Start Test Mode>Step to preview results to get the optimum identification).
- Add another IdentifyPrimaryObjects module, this time for identification of microcompartments. Change the name of the primary objects e.g. Microcompartments, keep settings the same as the previous step but change the typical diameter and threshold values to match the smaller, brighter objects.
- Add another module by navigating Add>All>MeasureObjectSizeShape. In ‘select object to measure’ select Cells. Click ‘Add another object’ and select Microcompartments.
- Add another module by navigating Add>All>ExportToSpreadsheet. Select column delimiter as “Tab” and select ‘output file location’ as ‘default output folder’ (make sure to set this correctly by clicking ‘view output settings’ and selecting the preferred output folder). Set the ‘Filename prefix’ e.g. “MyExpt_”. Select ‘Yes’ to ‘Select the measurements to export’ and press the button to select measurements. Here you can select which data you want to be exported, in the case of determining circularity of microcompartments, navigate All>Microcompartments>AreaShape>Eccentricity and tick, and also tick All>Microcompartments>Number. In this example an Excel file will be generated with data about circularity of microcompartments which allows them to be ranked by how properly they have formed.
- After all modules have been configured and a default output folder selected, click on ‘Analyze Images’ to perform analysis and output data.
-This covers the analysis modules for this simple protocol but a more in-depth, custom analysis of data is possible. Different data can be exported to excel, correlation of different fluorescence tags can be analysed, and images with object outlines can be exported (see links above).
Materials (consumables):
Fixed cell suspension
0.3% Triton-X-100
PBS
DAPI (2mg/ml)
Blank microscope slides
Cover slips
Method:
- Follow steps 1-8 of the Cell Fixation (using paraformaldehyde) protocol.
- Make the cells permeable by treating them with 100µl of 0.3% (v/v) Triton-X-100 solution for 5 minutes.
- Spin down at 5000rpm for 2 minutes in a table top centrifuge, discard the supernatant and resuspend in 50µl PBS.
- Add 50µl of DAPI to the eppendorf and leave for 30 minutes.
- Spin down at 5000rpm for 2 minutes, discard the supernatant and resuspend in 50µl PBS.
- Pipette 12μl of suspension to a slide, add a cover slip and allow to dry in the dark.
- Observe the slides under confocal microscope by setting the excitation filter at 370nm and emission filter at 526nm.
(All the further steps must be carried out in the dark.)
(Adapted from the protocol found here).
Materials (consumables):
Dye MasterMix: 0.12% Syro Orange Dye in HEPES buffer (pH 7.2)
Protein Solutions (concentration varies depending on protein - we recommend an optimisation stage is conducted prior to the full protocol being run)
Methods:
- Add 30uL of dye mastermix to each well (in a 96-well plate).
- Add 10uL of protein solution to the corresponding wells, and 10uL buffer into the control wells.
- Run reaction in a CFX connect real-time system from 25C to 95C, at 0.4C intervals. Take measurements after 30secs at each temperature.
Materials:
Ni-NTA agarose affinity media
Plastic gravity flow column with porous polyethylene filters and stopper
Cell pellet containing his-tagged recombinant protein
10% SDS-PAGE gel, power source and appropriate visualization equipment
Rollers (in at 4C cabinet)
Stirring plate
Dialysis tubing and clips
VivaSpin columns with appropriate molecular weight cut off
Lysis Buffer pH 8 (250mM NaCl, 25mM Tris, 10mM Imidazole, 5mg/L Lysozyme, 5mg/L DNaseI, Roche Complete Protease Inhibitor)
Wash Buffer 1 pH 8 (250mM NaCl, 25mM Tris, 10mM Imidazole)
Wash Buffer 2 pH 8 (250mM NaCl, 25mM Tris, 50mM Imidazole)
Elution Buffer pH 8 (250mM NaCl, 25mM Tris, 200mM Imidazole)
Methods:
- Clean affinity media: Ni-NTA agarose can be stored in 20% ethanol to prolong its shelf life, wash away the ethanol by pipetting the Ni-NTA agarose slurry into a plastic gravity flow column and washing with 10 bed volumes of distilled water. Then equilibrate with 5 bed volumes of wash buffer. Cap the column and keep the affinity media in wash buffer 1 until use
- Pre-chill all buffers. Prepare Lysis buffer on the day it is to be used.
- Transfer cell pellet to a closed container with 5ml of pre-chilled lysis buffer per gram of cell pellet. Allow to thaw on rollers at 4°C for 30 minutes.
- Disrupt cells by sonication on ice.
- Separate cell debris from soluble protein by centrifugation at 10,000 rpm for 30 minutes at 4°C. Transfer supernatant to a fresh tube.
- Resuspend the Ni-NTA agarose into a slurry in the wash buffer 1 by pipetting up and down.
- Add 1 ml of Ni-NTA slurry to the lysate supernatant. Note that for large volumes of supernatant, a larger volume of slurry will be needed. We used an excess of 1 ml of slurry for every 50 ml of supernatant. Mix the slurry and lysate on rollers for >30 minutes in rollers at 4°C.
- Load the lysate into a plastic gravity flow column at 4°C and collect flow through. Keep all fractions on ice.
- Wash the resin with 10 bed volumes of wash buffer 1, then wash buffer 2, collect fractions.
- Elute the protein with 5 bed volumes of elution buffer.
- Analyse fractions by SDS-PAGE.
- Transfer fraction containing the recombinant protein to dialysis tubing and dialyse into an excessive volume of the desired buffer at 4°C with gentle stirring. Replace the buffer after 45 minutes, then repeat.
- Concentrate the purified protein using a VivaSpin column according to the manufacturers’ instructions.
- Measure the absorbance at 280 nm of the purified protein, and calculate the final concentration using the proteins’ extinction coefficient. This can be calculated by inputting the proteins’ primary sequence into a tool like ProtParam from ExPASy.
- Add Glycerol to a final concentration of 5% v/v and flash freeze in liquid nitrogen prior to storage at -80°C.
Materials:
OneTaq Mix
Primers
Template DNA
ddH20
Methods:
Reaction set up:
| Component | Concentration |
|---|---|
| Fwd Primer | 10uM |
| Rev Primer | 10uM |
| 2X OneTaq Mix | 1X |
| Nuclease-free water | to 10µl |
Thermocycling conditions:
| Step | Temperature | Time |
|---|---|---|
| Initial Denaturing | 95C | 30sec |
| 30 cycles: | 95C | 15-30sec |
| ~55C | 15-60sec | |
| 68C | 1min/kb | |
| Final Extension | 68C | 5min |
| Hold | 4-10C | inf |
Materials:
DNA fragments
Gibson assembly master mix
dH20
Methods:
- Set up the following reaction on ice:
2-3 Fragment Assembly 4-6 Fragment Assembly Positive Control Total Amount of Fragments: 0.02-0.5 pmols 0.2-1 pmols 10uL Gibson assembly master mix (2X): 10uL 10uL 10uL Deionized water: to 20uL to 20uL to 20uL - Incubate samples in a hermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
