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Materials

5x Isothermal Reaction Mix (6mL total)

Assembly Master Mix (1.2mL total)


Method

  1. Thaw a 15 µL assembly mixture aliquot and keep on ice until ready to be used
  2. Add the DNA fragments to assemble to the mixture. The total volume of DNA to be added is 5 µL for a total of 20 µL reaction volume. Typical amounts of DNA are 10-50 fmol of each fragment
    • When assembling DNA fragments to form a new plasmid, a two to three times excess of insert DNA relative to the plasmid backbone is desirable. If the insert is very small, increase its concentration relative to the backbone to a five times excess - otherwise they risk being entirely digested by the exonuclease
  3. Incubate at 50ºC for 15 to 60 min (60 min is optimal)
  4. Transform 10 µL of the reaction volume as usual. Freeze the other 10 µL as back up

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Materials

E. Coli culture

5mL of LB media in a 50mL falcon tube

50mL of LB media in a flask

20mL of 0.1 M CaCl2

10mL of 0.1 M CaCl2, 15% glycerol v/v


Methods

Overnight culture

  1. Inoculate 5mL of LB media in a 50 mL falcon tube - with a single E.coli colony
  2. Incubate in a shaking incubator for approximately 16-18 hours, eg. overnight.
    • Incubation temperatures and RPM will vary based on the strain and incubator, check information on the strain to determine optimal growth conditions.
  3. Centrifuge cells at ~3000rcrf for 5 minutes or until the cells have formed a pellet.
  4. Remove supernatant and resuspend in 5mL of ether sterile PBS or fresh sterile media.
    • This step is more important if you are preparing competent cells from a strain already containing a plasmid with antibiotics.

Day culture and preparation of competent cells

  1. Inoculate the 50mL flask of LB media with 0.5mL of resuspended overnight culture from the previous step above.
  2. Incubate flask in a shaking incubator at appropriate temperature and RPM. Allow the culture to grow to an OD600 of 0.4-0.6.
    • Common strains will take around 2-3 hours to reach this OD600.
  3. Once at the appropriate OD600 pour cells into a sterile 50mL falcon tube and place in ice for 10 minutes.
    • All work from this step forward should be performed on ice
    • Solutions and incubators should be prechilled (including inserts) to 4°C
  4. Centrifuge cells at ~3000rcrf for 5 minutes or until the cells have formed a pellet.
  5. Discard supernatant.
  6. Resuspend cells in 20mL of 0.1 M CaCl2
  7. Leave resuspended cells on ice for 1 hour in 0.1 M CaCl2
  8. Centrifuge cells at ~3000rcrf for 5 minutes or until the cells have formed a pellet.
  9. Discard supernatant.
  10. Resuspend cells in 10mL of 0.1 M CaCl2, 15% glycerol
    • Cells can be used for immediate transformation
  11. If storing competent cells for use later aliquot 200-400μL of cells into sterile tubes.
    • Once competent cells have been defrosted they can not be stored away again
  12. Place aliquots in a -80°C freezer.

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Materials

Lysis buffer
LB Agar plates with ampicillin
PCR reagents
Pipette tips


Method

Prepare colony patches

  1. Prepare LB Agar + relevant antibiotic
  2. Write numbers on the plate or draw a grid, number each grid square.
    • 20 to 30 numbers should fit depending on the desired size of patches
  3. Pick a colony using a pipette tip or toothpick from the plate you wish to screen. Patch it (make a small streak) over the first number/ square on the new plate.
  4. Change the tip and patch the next colony. Patch as many as necessary.
    • If screening for a successful Gibson assembly, 10 colonies are sufficient.
  5. Let the patches grow overnight.

Cell lysis for cPCR

  1. Label as many 1.5 mL tubes as colonies you wish to screen
  2. Add 15 µL of lysis buffer to each tube
  3. Touch the first patch with a pipette tip (as vertically as possible) to transfer cells from the patch to the tip
  4. Place the tip in the first tube
  5. Repeat with as many patches as planned
  6. Give each tip a swirl to fully release the cells from the tip into the lysis buffer
  7. Close the tubes and boil the samples at 100 ºC for 15 minutes
  8. Centrifuge the tubes for 10 minutes at maximum speed (ca. 13, 000g) in a benchtop centrifuge. The supernatant contains the plasmid DNA

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Introduction

This protocol is designed for the purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of E.coli in LB medium.

Note: All protocol steps should be carried out at room temperature.


Materials

Pelleted Bacterial Cells
250uL Buffer P1 (Resuspension Buffer)
250mg RNase A

250uL Buffer P2 (Lysis Buffer)
Buffer P3 (Neutralisation Buffer)
Buffer N3 (Neutralisation Buffer)
Buffer PE (Wash Buffer)
QIAprep 2.0 Spin Column
Buffer EB (Elution Buffer)


Methods

  1. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube. Ensure that RNase A has been added to Buffer P1
    • No cell clumps should be visible after resuspension of the pellet
  2. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times
    • Mix gently by inverting the tube. Do not vortex, because this will result in shearing of genomic DNA and contamination of plasmid.
    • Continue inverting the tube until the solution becomes viscous and slightly clear.
    • Do not allow the lysis reaction to proceed for more than 5 min.
  3. Add 350 μl Buffer N3. Mix immediately and thoroughly by inverting the tube 4–6 times
    • To avoid localized precipitation. The solution should become cloudy.
  4. Centrifuge for 10 min at ~17,900g in a microcentrifuge
    • A compact white pellet will form
  5. Apply 800 μl of the supernatant from step 4 to the QIAprep 2.0 Spin Column by pipetting
  6. Centrifuge for 30–60s. Discard the flow through.
  7. Wash QIAprep 2.0 Spin Column by adding 0.75 ml Buffer PE and centrifuging for 30–60 seconds
  8. Discard the flow through
  9. Centrifuge at full speed for 1 min to remove residual wash buffer
    • Important: Residual wash buffer will not be completely removed unless the flow through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.
  10. Place the QIAprep 2.0 Spin Column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep 2.0 Spin Column, let stand for 1 min, and centrifuge for 1 min

Reference

QIAGEN (2020). QIAprep Miniprep Handbook. QIAGEN, QIAGEN.


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Introduction

This protocol has been written specifically with the strain T7 express in mind and the plasmid pET-19b. However it can be adapted to other strains via optimisation.


Materials

E. Coli strain for protein expression

LB Media

Sterile PBS

100mL LB media in a 500mL flask Appropriate antibiotic stocks

Induction agent stock

Methods

Overnight Culture

  1. Add 5mL of LB media and the appropriate amount of antibiotics to a 50mL falcon tube.
  2. Inoculate the 50mL falcon tube from step 1 with a single colony from an agar plate
  3. Incubate the tube in a shaking incubator at 37°C for 16 hours
    • The RPM will be dependant on the incubator

Day Culture

  1. Centrifuge tubes at 3000rcf for 10 minutes or until the media is clear and a pellet has been formed
  2. Discard supernatant
  3. Resuspend pellet in 5mL of sterile PBS
  4. Add appropriate antibiotics to the 100mL of LB media in a flask
  5. Inoculate the flask with 1mL of culture with the tube from step 3
  6. Incubate the flask in a shaking incubator set at 37°C, until the culture reaches on OD600 of 0.4-0.6
    • It will normally take around 3-4 hours for the culture to reach 0.4-0.6 OD600
  7. Once the culture has reached an OD600 of 0.4 to 0.6 add the appropriate amount of induction agent
  8. Incubate the flask in a shaking incubator
    • Time/temperature varies for each individual strain/plasmid combination
    • If using T7 express a 3-4 hour incubation at 37°C is the best place to start

Harvest

  1. Pour culture into tubes and centrifuge at 3000rcf for 10 minutes or until the media is clear and a pellet has been formed
  2. Discard supernatant
  3. Resuspend pellets in PBS and combine all pellets that were from the same culture into one tube
    • Weigh the tube before adding anything to it so that the size of the pellet can be determined
  4. Centrifuge at 3000rcf for 10 minutes or until the media is clear and a pellet has been formed
  5. Discard supernatant and store the tube at -20°C
    • Weigh the tube before storing it and note the size of the pellet

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Introduction

This protocol is primarily a summary of the protocol provided with the BugBuster Master Mix. The samples used should have been acquired while performing protein expression. Knowing the OD600 of the cultures at the time the sample was taken is essential. If this information was not noted it may be possible to weigh the pellets and use that information to normalize the concentrations of the samples.


Materials

1 Sample of culture - Pre-Induction
1 Sample of culture - Post-Induction
Bug Buster Master Mix


Methods

  1. Pellet cells by centrifuging at 13,000g for 5 minutes
  2. Discard all supernatant
  3. Resuspend cells in _μL of BugBuster Master Mix
    • A 100μL sample with an OD600 of 1.0 (at the time of the sample being taken) should be resuspended in 100μL. This will ensure the concentration of protein in each samples is consistent
  4. Incubate at room temperature for 20 minutes
  5. Centrifuge samples at 16,000g for 20 minutes
  6. Transfer supernatant to a new tube
    • This supernatant is your soluble protein do not throw it away
  7. Resuspend the pellet, from the original tube, in 1mL of PBS buffer
  8. Centrifuge samples at 10,000g for 10 minutes
  9. Discard supernatant
  10. Resuspend the pellet in 1mL of PBS buffer
  11. Centrifuge samples at 5,000g for 10 minutes
  12. Discard supernatant
  13. Resuspend the pellet in a volume equal to that used in step 3 with PBS buffer
    • This sample is your insoluble protein
  14. Load 15μL of each sample on an SDS-PAGE gel
    • There should be 4 samples per culture, an insoluble and soluble of both the initial pre induction, and the post induction samples

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Materials

Poly-Prep Chromatography Column
HisPur Ni-NTA Resin
Elution Buffer

5CV Wash Buffer
5CV Binding Buffer


Method

  1. Pack the column by loading 1mL of Slurry
    • Slurry is made of equal parts by volume resin and 20% ethanol
  2. Allow 20% ethanol to flow through
  3. Equilibrate column by adding 5CV of binding buffer and allow to flow through. Repeat twice
    • If a column contains 1mL of resin the column volume (CV) is 1mL
  4. Load clarified lysate onto column. Allow it to flow through
    • Clarified lysate will be made by: lysing the (previously sonicated) pellet in the binding buffer, then centrifuging (10,000gx10mins) and filtering it (0.45 µm pore size, PES/PVDF)
    • This will remove all cell debris and insoluble protein. If the protein of interest is insoluble modifications will need to be made to this process
    • The flowthrough coming out of the column should be saved for later analysis
  5. Load 5 CV of binding buffer
    • Removes any unbound protein still found on the column
  6. Load 5 CV of elution buffer
    • Collect Elution Fractions in separate tubes
    • Majority of proteins will be eluted in the first elution fractions
  7. Load protein purification fractions on an SDS-PAGE gel – for visualisation

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Materials

Binding Buffer
3.5mL Protein 1 - attached to mVenus Fluorophore
2mL Protein 2 - attached to mCerulean Fluorophore
2mL mVenus Fluorophore (pure)
1mL mCerulean Fluorophore (pure)
PBS


Method

Cell lysis via sonification

  1. Re-suspend cell pellet in Binding Buffer
  2. Sonicate cells for 8 minutes at 2 second intervals and 40% amplitude. Keep on ice while sonicating
  3. Centrifuge cell lysate for 30 minutes at 10,000 x g to seperate soluble protein from cell debris/insoluble protein

Loading onto 96 well plate

  1. Add 200uL of TaISP-mVenus protein to Well 1 of a 96-well plate
  2. Add 800uL of Pst_12806-mCerulean protein to the same well as step 1
  3. Create a series of serial dilutions for the following 8 wells, at a dilution factor of 4:5
    • E.g. transfer 800uL from well 1 to well 2. Following this add 200uL of TaISP-mVenus protein. Final volume of the well should be 1000uL
  4. Repeat steps 1-3, to create the assay in duplicate row
  5. In a new third row, add 200uL of mVenus protein to well 1
  6. Add 800uL of mCerulean protein to the same well as step 5
  7. Create a series of serial dilutions for the following 8 wells, at a dilution factor of 4:5
    • This serves as a negative control
  8. Analyse each well using NanoDrop, to standardised protein concentrations across wells. This is done by adding PBS to dilute the sample
    • Due to inconsistencies in pipetting
  9. The 96 well plate is now ready for FRET analysis

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Making an agarose gel (1%)

Materials

Agarose powder
1x TAE buffer
Microwavable flask


Method

  1. Using a scale, weigh out 0.5g of agarose powder and transfer it into a microwavable flask
  2. Pour 50 mL of 1x TAE buffer into the flask and swirl to mix. A dye may be added at this stage
  3. Using a microwave to heat up the mixture, make sure all of the powder dissolves
  4. Pour the mixture into a casting tray, then add your well combs and wait for it to set

Running an agarose gel

Materials

Gel electrophoresis chamber
Power source
Cables


Method

  1. Once the gel is set and in the gel electrophoresis chamber, pour 1x TAE buffer until it covers the gel, then load your samples
  2. Connect the positive nodes of the power source to the positive node of the chamber and the negative nodes to each other
  3. Run the gel at 110V for 45 minutes or until the DNA has run far enough
  4. Then take the gel out for imaging and analysis after switching off the power supply

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Making the Bis-Tris PAGE Gel (10%) Separation Gel

NOTE: Method is under "Making the Bis-Tris PAGE Gel (4%) Stacking Gel"


Materials

2.3 mL Water
3x 2.33mL Bis-Tris (1M)
2 µL TEMED
35 µL APS (10%)
2.33 mL Acrylamide:Bis (37.5:1) (30%)
Isopropanol
15 mL Falcon tube
Gel Casting Glass Plates (front and back plate with rubber stopper in between)
Gel Casting Chamber


Making the Bis-Tris PAGE Gel (4%) Stacking Gel

Materials

1.05 mL Water
3x 0.67 mL Bis-Tris (1M)
2 µL TEMED
10 µL APS (10%)
270 µL Acrylamide:Bis (37.5:1) (30%)
15 mL Falcon tube


Method

  1. Make the separation gel by mixing all the components in a 15 mL Falcon tube, making sure to add in the acrylamide:bis last as that begins the soldification of the gel
  2. Form the gel casting chamber by placing the front and back plate with the rubber stopper in between inside
  3. Pour the solution in between the glass plates
  4. Apply a small amount of isopropanol to the surface to ensure the gel is even when set
  5. Make the stacking gel by mixing all the components in a 15 mL Falcon tube making sure to add in the acrylamide:bis last as that begins the soldification of the gel
  6. Once the separation gel has set, pour the stacking gel on top and add your well comb and wait for it to set

Running Bis-Tris PAGE Gel

Materials

Gel tank
Power source
Cables
1x Bolt buffer


Method

  1. Once the gel is set, remove the well combs and place it into a protein gel tank
  2. Fill the tank up to the indicated line with 1x Bolt buffer
  3. Load the samples into the wells
  4. Connect the positive nodes of the power source to the positive node of the chamber and the negative nodes to each other
  5. Run the gel at 110V for 45 minutes or until the protein has run far enough
  6. Take the gel out for imaging and analysis after switching off the power supply

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