Team:UC Davis/Experiments

Cyantific: UC Davis iGEM 2016


Protocols

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Gel Electrophoresis

Agarose Gel Recipe and Running Gel Electrophoresis Goal: Size select to make sure you have correct DNA sequence amplified

Material Quantity
Agarose 0.5g
1X TAE 50ml
SYBR safe gel stain 3uL
Tube of 6x Loading Dye 1
Strip of Parafilm 6”
80-Well Tube Rack 1
PCR Tube Rack 1
  1. Make the Gel:
    1. Measure 0.5 g of agarose.
    2. Add 50 mL of 1X TAE (Tris Base, Acetic Acid, EDTA) Buffer.
    3. Microwave for ~1 minute or until agarose is melted.
    4. Cool to ~45°C, or warm to the touch.
    5. Add 3 μL of SYBR Safe gel stain (this is an intercalating dye that fluoresces when DNA is present) and mix well without producing bubbles.
    6. Pour melted agarose into a gel mold with a comb in place.
    7. Pop any lingering bubbles with a pipette tip.
    8. Cure agarose gel at room temperature.
  2. Run the Gel:
    1. Stretch the piece of parafilm across one end of the 80-well tube rack. Make wells in the parafilm by pushing gently into the parafilm.
    2. Pipette out 1.5 μL of the 6X loading dye for each of your wells.
    3. Add 5 μL of your PCR reaction to each well and mix by gently pipetting up and down. (Make sure to keep track of which sample is which)
    4. Add 5 μL of each sample into the gel. Ensure that there is at least one lane with 5 μL of ladder/standard.
    5. Run the Gel at 120 V for 30 minutes.

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Transformation Protocol

Goal: Get DNA sequence of interest into E.Coli Cells

Material Quantity (per sample)
Competent Cells 50 µL
Luria Broth 800 µL
  1. Label 14 mL Falcon tubes and place them on ice.
  2. Gently thaw tube of competent cells in the palm of the hand. Once the cells have thawed, IMMEDIATELY transfer the tube on ice.
  3. Pipette 50 µL of competent cells into the bottom of each Falcon tube
  4. While gently swirling to mix, add 7.5 µL of transforming DNA into the small pool of competent cells on the bottom of each correspondingly labeled tube.
  5. Incubate the cells on ice for 15 mins.
  6. Bring samples to the 42°C water bath. Heat shock for exactly 45 seconds.
  7. Immediately place tubes back on ice for 1-2 mins.
  8. Working sterilely, add 800 µL of LB to each tube.
  9. Place your tubes into a 37°C incubator shaker for 45-60 mins.
  10. Label your plates accordingly
  11. Plate 200 µL of your transformed cells on each corresponding plate.
    • Use glass beads to spread your cells evenly.
  12. Place in static 37°C incubator for ~16 hours.

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Protein Purification Protocol

Goal: Purify the CBCR protein from cells

  1. Expression
    1. 37degC → Shake 2-6 hours
    2. 24degC → Shake 12 - 25 hours
    3. 18degC → 16 - 36 hours
    4. Transfer cells to 50mL Falcon and spin for 20-40min at max speed
    5. Decant supernatent and store cells in -20
    6. Initiate cultures in 50mL TB + Antibiotic (i.e. rich and buffered media) using an inoculum from an overnight or a fresh colony and shake at 37degC
    7. Measure OD every 0.5-2 hours (i.e. take 100uL and add to 1mL) until OD reaches 1 (for blue… maybe higher for red?) in the culture (so would read 0.1 in instrument since there was a 10x dilution).
    8. Add inducing agent and shake at defined temperature
    9. Harvest cells
  2. Lysis
    1. Vortexing is fine, once resuspended keep on ice
    2. Save 10uL for gel analysis!
    3. Sonicate the cell suspension with 10 short burst of 10 sec followed by intervals of 30 sec for cooling. Keep the suspension at all times in water-ice bath. (Avoid foaming)
    4. Remove cell debris by ultracentrifugation at 4°C for 30 min at 45 000 rpm using a 45Ti rotor
    5. Transfer the supernatant to a fresh tube-- this is your crude protein extract
    6. Save 10uL for gel analysis before next step
    7. Resuspend cells in 0.5mL of lysis buffer
    8. Sonication
  3. Purification
    1. For every 1mL of culture, add 200uL of bead slurry (ie 20mL for every 1L culture) ~ for example 50mL culture = 1mL bead slurry
    2. In a fresh 2mL tube aliquot bead slurry of chitin binding resin
    3. Spin down at 1000rpm for 1min and pippette off ethanol supernatent
    4. Resuspend in wash buffer
    5. Spin down at 1000rpm for 1min and pippette off supernatent
    6. Add Supernatent from lysis
    7. Incubate on rocker in cold room for 2 hours
    8. Spin down at 1000rpm for 1min and pippette off supernatent
    9. Save supernatent as this is your flow through and will want to analyze on a gel
    10. Resuspend in 1mL wash buffer
    11. Transfer to a 50mL Falcon tube with 10mL of wash buffer
    12. Incubate on rocker in cold room for 0.5 hours
    13. Spin down at 1000rpm for 5min and decant/pippette off supernatent
    14. Add 10mL of wash buffer
    15. Incubate on rocker in cold room for 0.5 hours
    16. Spin down at 1000rpm for 5min and decant/pippette off supernatent
    17. Add 1mL of cleavege buffer
    18. Transfer to 2mL tube & incubate overnight (16-24 hours) at room temperature (or potentially cold room… depends on stability of protein) on rocker
    19. Spin down at 1000rpm for 1min
    20. Transfer supernatent to fresh tube, this should be your protein!
    21. Transfer Chitin resin to “USED” tube that is in 50% Ethanol at 4deg. Save until significant amount of material and then regenerate as described below.
    22. Analyze Purified protein (Spectrum and SDS Protein Gels)
    23. OPTIONAL: May need to concentrate and/or buffer exchange using a concentrator.
    24. ***GIVEN THE LOW BINDING CAPACITY OF THE COLUMN IT IS LIKELY YOU WILL NEED TO CONCENTRATE FOR WHAT WE ARE DOING*** Make sure to talk a lot of Wilson, Katie, Youtian in the Siegel group who do protein production and purification on an almost daily basis (show em this protocol as we generally use his beads with his tagged proteins which have a ~10x higher binding capacity).

https://www.thermofisher.com/us/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/concentrating-cells.html

http://wolfson.huji.ac.il/purification/PDF/Protein_Expression_Extraction/NOVAGEN_BugBuster_protein_extraction.pdf

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PCR Protocol 50 uL Reaction

Goal: To amplify DNA given

Materials for a 50 μL reaction

MaterialQuantity
10 uM Forward Primers 2.5 uL
10 uM Reverse Primers 2.5 uL
1 ng/µL DNA template, 10 ng/uL DNA template 10 uL,1ul + 9 ul H20
5x HF Reaction Buffer 10 uL
10 mM dNTPs 1 uL
Phusion HF Polymerase 0.5 uL
MilliQ Water 23.25 uL

Ensure reagents are thawed, mix well and spin down. Add 10 ng of appropriate template to each PCR tube Add 2.5 µL of correctly paired forward and reverse primers to each PCR tube Add water then 5X buffer then dNTPs to each tube Remove Phusion from freezer and add to each PCR tube. Phusion is temperature sensitive: keep cool. Mix well and spin down. Place tubes in PCR machine: use Phusion protocol in the BIM143L folder. Ensure that the extension time is correct for the size of the DNA fragment you are amplifying. Phusion polymerase has an extension rate of 1kb/30sec.

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PCR Purification

Goal: Purify your amplified DNA sequence.

MaterialQuantity (per sample)
NucleoSpin® Gel and PCR Clean-up Column 1
Buffer NTI 2 volumes per volume of sample
Buffer NT3 700 μL
MiliQ H2O OR Buffer NE 20 μL OR 15-30 μL
  1. Mix 1 volume of sample to 2 volumes of Buffer NTI
  2. Place a NucleoSpin® Gel and PCR Clean-up Column into a Collection Tube (2 mL) and load up to 700 μL sample
  3. Centrifuge for 30 s at 11,000 x g. Discard flow-through and place the column back into the collection tube.
    • Load remaining sample if necessary and repeat the centrifugation step.
  4. Add 700 μL Buffer NT3 to the NucleoSpin® Gel and PCR Clean-up Column. Centrifuge for 30 s at 11,000 x g. Discard flow-through and place the column back into the collection tube
  5. Centrifuge for 1 min at 11,000 x g to remove Buffer NT3 completely. Make sure the spin column does not come in contact with the flow-through while removing it from the centrifuge and the collection tube.
  6. Place the NucleoSpin® Gel and PCR Clean-up Column into a new 1.5 mL microcentrifuge tube (not provided). Add 15–30 μL Buffer NE and incubate at room temperature (18–25 °C) for 1 min. Centrifuge for 1 min at 11,000 x g. OR add 20 uL of water and centrifuge for 10 min
  7. Dispose of the column, keep the flow-through.

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Oligo Annealing

  1. Add 10uL of each complementary oligo in one PCR tube (100uM)
  2. 95°C for 2 min, ramp down to 25°C over 45 min, and then hold at 4°C

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Mini Prep

Goal: Extract correct plasmid out of colony so you can transform more E.Coli cells Harvest Centrifuge the overnight LB-culture. (1min 4000RPM) Remove all medium. Resuspend Add 250 μL Resuspension Buffer (R3) with RNase A to the cell pellet and resuspend the pellet until it is homogeneous. Transfer to new 1.5 mL microcentrifuge tube. Lyse Add 250 μL Lysis Buffer (L7). Mix gently by inverting the capped tube until the mixture is homogeneous. Do not vortex. Incubate the tube at room temperature for 5 minutes. Precipitate Add 350 μL Precipitation Buffer (N4). Mix immediately by inverting the tube, or for large pellets, vigorously shaking the tube, until the mixture is homogeneous. Do not vortex. Centrifuge the lysate at >12,000 × g for 10 minutes. Bind Load the supernatant from step 4 onto a spin column in a 2-mL wash tube. Centrifuge the column at 12,000 × g for 1 minute. Discard the flow-through and place the column back into the wash tube. Wash and remove ethanol. Add 700 μL Wash Buffer (W9) with ethanol to the column. Centrifuge the column at 12,000 × g for 1 minute. Discard the flow-through and place the column into the wash tube. Centrifuge the column at 12,000 × g for 1 minute. Discard the wash tube with the flow-through. Elute Place the Spin Column in a clean 1.5-mL recovery tube. Add 50 μL of ultrapure H2O to the center of the column. Incubate the column for 5 min at room temperature. Recover Centrifuge the column at 12,000 × g for 2 minutes. The recovery tube contains the purified plasmid DNA. Discard the column. Store plasmid DNA at 4°C (short-term) or store the DNA in aliquots at −20°C (long-term).

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Golden Gate Assembly

  1. Set up assembly reactions:
Material (20 µL Reaction) Quantity (per sample)
10X Ligase buffer 2 µL
DNA Parts 40 fmol
100X BSA 0.2 µL
BsaI (20U/uL) 0.75 µL
T4 ligase (400U/uL) 1 µL
H20 Less than 20 µL
  1. Thermocycler protocols:
    • Engler 2011: (37C for 2 min, 16oC for 3 min)x50, 50C for 5 min, 80C for 5 min

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Gel Purification

Goal: Purify your amplified DNA sequence from a gel extract.

MaterialQuantity (per sample)
NucleoSpin® Gel and PCR Clean-up Column 1
Buffer NTI 2 µL per mg of gel (if <2% agarose)
Buffer NT3 700 μL
MiliQ H2O OR Buffer NE 20 μL OR 15-30 μL
  1. Excise DNA fragment / solubilize gel slice
    • Note: Minimize UV exposure time to avoid damaging the DNA.
  2. Take a clean scalpel to excise the DNA fragment from an agarose gel. Remove all excess agarose. Determine the weight of the gel slice and transfer it to a clean tube. For each 100 mg of agarose gel < 2 % add 200 μL Buffer NTI. For gels containing > 2 % agarose, double the volume of Buffer NTI. Incubate sample for 5–10 min at 50 °C. Vortex the sample briefly every 2–3 min until the gel slice is completely dissolved!

    Bind DNA
  3. Place a NucleoSpin® Gel and PCR Clean-up Column into a Collection Tube (2 mL) and load up to 700 μL sample. Centrifuge for 30 s at 11,000 x g. Discard flow-through and place the column back into the collection tube. Load remaining sample if necessary and repeat the centrifugation step

    Wash silica membrane
  4. Add 700 μL Buffer NT3 to the NucleoSpin® Gel and PCR Clean-up Column. Centrifuge for 30 s at 11,000 x g. Discard flow-through and place the column back into the collection tube. Dry silica membrane
  5. Centrifuge for 1 min at 11,000 x g to remove Buffer NT3 completely. Make sure the spin column does not come in contact with the flow-through while removing it from the centrifuge and the collection tube.

    Elute DNA
  6. Place the NucleoSpin® Gel and PCR Clean-up Column into a new 1.5 mL microcentrifuge tube (not provided). Add 15–30 μL Buffer NE and incubate at room temperature (18–25 °C) for 1 min. Centrifuge for 1 min at 11,000 x g OR add 20 uL of water and centrifuge for 10 min

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Preparing Freezer Stocks

  1. Obtain cryo storage tubes, label them clearly
  2. Mix 800 µL of liquid culture (previously grown to high density) with 800 µL of 80% glycerol stock
  3. Pipet up and down intensely yet gently to mix them thoroughly
  4. Place tubes into MPBIL storage box in -80 freezer
  5. Update registry in Box (the virtual one, not the physical one)

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Colony PCR

Goal: Amplify DNA of colony so you know colony transformed correctly/as expected

MaterialQuantity
10 µM Forward Primers 1 µL
10 uM Reverse Primers 1 µL
Q Solution 4 µL
10X Buffer 2 µL
10 mM dNTPs .5 µL
TAQ Polymerase 0.2 µL
MilliQ Water 11.3 µL
  1. Use a sharpie to mark 2 colonies that will screen the transformation plate. Label colonies with initials.
  2. On replicate plate, mark 2 locations where you will put the replicate colonies. Label each spot in the same way as from the transformation plate.
  3. Pipette 10 uL of sterile H2O into 2 PCR tubes
  4. Use 10 uL pipette tip to pick up colony (gently touch side of tip to colony), gently touch marked location on replicate plate and put the tip into one of the PCR tubes
  5. Repeat for the other colony
  6. Calculate master mix for two PCR reactions
  7. In 1.5 mL microcentrifuge tube, make master mix. Add TAQ last. Mix and spin down.
  8. Attach one of the tips in the PCR tube to a 10 uL pipette. Slowly pipette up and down to empty out cells, and then discard tip.
  9. Repeat for other tip.
  10. Add 10 uL of master mix to each PCR tubes
  11. Mix, spin down, and put in thermocycler.

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Chitin binding resin re-use

Goal: clean bead of any previous protein so it can be used again

  1. Take empty column and add10mL 0.3NaOH, 5 times at room temp.
  2. Neutralize flow through NaOH with HCl
  3. Allow to soak for 30 minutes
  4. Repeat with 10mL water, 20 times
  5. Repeat with 10mL wash buffer, 5 times
  6. Continue to step 4 of above protocol

Storing the purified protein:

  1. In the form straight from purification with 10% glycerol you need to freeze it using liquid nitrogen so that ice crystals don’t form and break the protein
  2. Otherwise, you need to make it ⅔ glycerol/ volume so that you can store it in a -20C

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Measuring Absorbance

Goal: Find dark state and photoproduct state absorbance

  1. Turn on, and allow spectrometer to warm up for ~15min
  2. Calibrate spectrometer with control solution of 50ul dialysis buffer
  3. Put 50ul purified protein into plate well (3 reps each protein)
  4. Take an absorbance spectrum ( 250-750nm - maybe try to narrow for more exact measurement once we know where it is)
  5. Take a laser pointer and shine light on sample for at least 1 minute or until the entire sample changes color
  6. Measure absorbance again (x3)
  7. Wait until color is back to dark state, shine light on it again until all changed, start timer, and record how long it takes to revert back to dark state for a ~rough~ dark reversion time.

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Transferring Colony to 4 mL Culture

  1. Pipette 4 mL of LB into labelled culture tubes
  2. Add 4-6 uL of antibiotic to each tube
  3. Use pipette tip or loop to take a small sample from colony on plate, transfer into tube
  4. Culture overnight in shaker and check the next day

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Contact us at: ucdigem@gmail.com