Team:ULaVerne Collab/Experiments

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Experiments

  1. Turn on electroporator and set to 1.8kV, 200 ohms and 25 µF.
  2. Place recovery SOC in 37°C incubator.
  3. Pre-warm LB-antibiotic plates at 37°C.
  4. Thaw DH5-alpha electrocompetent cells on ice until the last ice crystals disappear.
  5. Place appropriate number of microcentrifuge tubes and 1 mm-electroporation cuvettes on ice.
  6. Gently flick the tube containing cells a few times to mix and add 20 µl to each of the microcentrifuge tubes.
  7. Add each DNA solution (in DI water) to the cells in each of the microcentrifuge tubes. Carefully stir with pipette tip to mix cells and DNA. Do not vortex. Leave on ice for 1 min.
  8. Transfer the DNA-cell mixture to the cold cuvette without introducing bubbles and make sure that the cells deposit across the bottom of the cuvette, tap on countertop 2X, wipe water from exterior of cuvette and place in the electroporation module and press pulse (don’t hold the button down). Time constant is around 4.8-5.1ms
  9. Immediately add 980 µl of 37°C SOC, mix by pipetting up and down once and transfer to a fresh microcentrifuge tube.
  10. Shake vigorously (250rpm) at 37°C for 1 h.
  11. Make appropriate dilutions. When using 10 ng of DNA, make two dilutions:
    Dilute 10 µl cells into 990 µl SOC and plate 100 µl. (1000-fold dilution)
    Dilute 100 µl cells into 900 µl SOC and plate 100 µl. (100-fold dilution)
    Also plate 100ul of undiluted cells (1x dilution)
  12. When spreading, dip the spreader into a beaker with ethanol to sterilize it. Run the spreader quickly through a flame to burn off the ethanol. Run the spreader on the empty plate to cool the spreader.
  13. Allow plates to dry with lids cracked, and incubate overnight at 37°C.
  1. Calculate how much LB media you will need by taking number of cultures x 4mL. Prepare LB media from powder by weighing 2.5g/100mL DI water. Add appropriate volume of 1000x antibiotic.
  2. Pipette 4mL of LB + antibiotic media into sterile 15mL conical tubes. One tube per colony.
  3. In one hand take a sterile pipette tip on the end of a pipette, with the other hand pick up the upside down plate containing the bacteria from the ligation. Turn the plate over in your hand so that the bacteria are now facing upwards towards you and touch the tip of the pipette tip gently to a bacterial colony that is completely isolated from any other colony.
  4. Now place the same tip with bacteria on it into one of the tubes containing LB media (from step 2) and move the tip around a bit to release some of the bacteria into the liquid. You may eject the pipette tip into the media but if you do this you will need to recover it the next day.
  5. Culture the tubes overnight in an incubated orbital shaker at 37ºC at 225 rpm.
  1. Transfer 1.9mL of overnight culture broth to 2.0mL labeled microcentrifuge tube.
  2. Spin tube at 8000rpm for 3 minutes at room temperature.
  3. Discard supernatant.
  4. Transfer 1.9mL of remaining overnight culture broth to the same tube.
  5. Spin tube at 8000rpm for 3 minutes at room temperature.
  6. Discard supernatant.
  7. Resuspend pellet in 250uL buffer P1 by pipetting up and down.
  8. Add 250uL buffer P2 and mix by inverting the tube 4-6 times until the solution becomes clear. Do not allow this reaction to proceed for more than 5 min.
  9. Add 350uL buffer N3 and mix by inverting the tube 4-6 times.
  10. Centrifuge for 10 min at 13,000 rpm at room temperature.
  11. Apply the supernatant to the top of a Qiaprep spin column (blue) that is fitted into a collection vial.
  12. Centrifuge the spin column for 1 min at 13,000 rpm at room temperature.
  13. Discard flow-through.
  14. Add 750uL Buffer PE to the top of the spin column.
  15. Centrifuge the spin column for 1 min at 13,000 rpm at room temperature.
  16. Discard flow-through.
  17. Centrifuge the empty spin column for 1 min at 13,000 rpm at room temperature to remove any residual buffer PE from the column.
  18. Place the spin column in a clean, labeled 1.7mL microcentrifuge.
  19. Add 50uL nuclease-free water to the center of the spin column.
  20. Centrifuge the spin column for 1 min at 13,000 rpm at room temperature.
  21. The purified DNA is now in the microcentrifuge tube and should be stored at -20deg C. Place the used spin column in a bottle of 1N HCl to recycle it.
Component 50 uL Reaction Final Concentration
Q5 High Fidelity 2X Master Mix 25 µl 1X
10 µM Forward Primer 2.5 µl 0.5 µM
10 µM Reverse Primer 2.5 µl 0.5 µM
Template DNA 1 ng/µl < 1000 ng
Nuclease Free Water to 50 uL

Transfer PCR tube to the thermocycler that is set with the following parameters:

STEP TEMP TIME
Initial Denaturation 98°C 30 seconds
30 cycles 98°C 5 seconds
*50–72°C 20 seconds
72°C 25 seconds/kb
Final Extension 72°C 2 minutes
Hold 4°C
Component 50 uL Reaction Final Concentration
Q5 High Fidelity 2X Master Mix 25 µl 1X
10 µM Forward Primer 2.5 µl 0.5 µM
10 µM Reverse Primer 2.5 µl 0.5 µM
Template DNA 1 ng/µl < 1000 ng
Nuclease Free Water to 50 uL

Transfer PCR tube to the thermocycler that is set with the following parameters:

STEP TEMP TIME
Initial Denaturation 98°C 30 seconds
30 cycles 98°C 5 seconds
*50–72°C 20 seconds
72°C 25 seconds/kb
Final Extension 72°C 2 minutes
Hold 4°C
  1. Excise the DNA band from the agarose gel with a clean, sharp scalpel. Use a 1.5 ml microfuge tube for processing up to 250 mg agarose per tube.
  2. Weigh the gel slice in a colorless tube. Then, Add Buffer QX1 according to DNA fragment size.
    (6 volumes for <100 bp; 3 volumes for 100 bp – 4 kb; 3 volumes with 2 volumes of water for >4 kb. Add 6 volumes of Buffer QX1 when using >2% or Metaphor agarose gels.) ex. if 100mg, three vol= 300uL
  3. Resuspend QIAEX II by vortexing for 30 s. Add QIAEX II to the sample and mix (vortex for ~ 30 sec):
    Use 10 μl QIAEX II for ≤2 μg DNA; 30 μl for 2–10 μg DNA; and an additional 30 μl for each additional 10 μg DNA.
    ***Determine by comparing band strength with bands of 1kb or APEX ladder
  4. Incubate at 50°C for 10 min to solubilize the agarose and bind the DNA.
  5. Mix by vortexing* every 2 min to keep QIAEX II in suspension. Check that the color of the mixture is yellow.
    If the color of the mixture is orange or violet, add 10 μl 3 M sodium acetate, pH 5.0, and mix.
    The color should turn to yellow. The incubation should then be continued for at least 5 min.
  6. Centrifuge the sample for 30 s and carefully remove supernatant with a pipet.
  7. Wash the pellet with 500 μl Buffer QX1. Resuspend the pellet by vortexing.*
  8. Centrifuge the sample for 30 s and remove all traces of supernatant with a pipet.
    This wash step removes residual agarose contaminants.
  9. Wash the pellet with 500 μl Buffer PE. Resuspend the pellet by vortexing.* Centrifuge the sample for 30 s and carefully remove all traces of supernatant with a pipet.
    Repeat once more. This step removes residual salt contaminants.
  10. Air-dry the pellet for 10–15 min or until the pellet becomes white. If 30 μL QIAEX II suspension is used, air-dry the pellet for approximately 30 min. Do not vacuum dry, as overdrying, may lead to decreased elution efficiency.
  11. To elute DNA, add 20 μl of 10 mM Tris·Cl, pH 8.5, TE buffer (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), or nuclease free water and resuspend the pellet by vortexing.
    * Incubate according to the DNA fragment size:
    5 min at room temperature (15–25°C) for ≤4 kb;
    5 min at 50°C for 4–10 kb;
    or 10 min at 50°C for >10 kb.
  12. Centrifuge for 30 s, and carefully pipet the supernatant into a clean tube.
  13. The supernatant now contains the purified DNA.

1. Set up the following reaction on ice:

Recommended Amount of Fragment Used for Assembly
2-3 Fragment Assembly 4-6 Fragment Assembly Positive Control
Recommended
DNA Ratio		 vector:insert = 1:2 vector:insert = 1:1
Total Amount of
Fragments		 0.03-0.2 pmols
x uL		 0.2-0.5 pmols
x uL		 10 uL
NEBuilder
HiFi DNA
Assembly Master
Mix		 10 uL 10 uL 10 uL
Deionized Water 10-X uL 10-X uL 0
Total Volume 20 uL 20 uL 20 uL
  • Assume you are starting with 50ng of vector. Calculate the volume needed for 50ng.
  • Use the NEB ligation calculator to calculate amount needed (in ngs) for each of your insert pieces.
  • Calculate the volume needed for each insert.
  • Calculate the amount of water you will need to add for a final volume of 20. If needed, you may increase the volume of the reaction to 30ul to accommodate more DNA pieces.
  • If this is done, then increase the volume of the Master Mix to 15ul.

    • 2. Incubate samples in a heat block 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.

    3. Transform NEB 5-alpha chemical competent E. coli cells with 5 μl or electro- competent cells with 2uL of the assembled product.

  1. After the tube was placed in the magnetic separator, remove the tube from the magnetic separator and add 500uL and mix it until its homogeneous.
  2. Place the tube in the magnetic separator and discard the supernatant.Note: Binding buffer: 50 mM NaH2P04, 300 mM NaCI, 20 mM imidazole, pH 8.0. Binding buffer: The choice of buffer depends on the particular properties of the protein. The buffer used most frequently is phosphate (50 mM is recommended). The pH of binding buffers generally leads to neutrality (7.0- 8.0). Note: to increase the selectivity of the binding of target protein it is necessary to add to the binding buffer a small concentration of imidazole (10-40 mM). We recommend 20mM as a guideline, but it will depend on each case. If the tagged protein doesn't bind under these conditions, the amount of imidazole should be reduced to 5-1OmM.
  3. Once the Renin is equilibrated the sample containing the fused protein for purification is applied. In some cases a slight increase of contact time may facilitate binding.Note: Binding capacity can be affected by several factors, such as sample concentration, binding buffer or contact time.Once the resin is equilibrated, add the clarified E coli lysate or protein extract. Mix the suspension gently for 30 min at room temperature or 1h at 4°C. In some cases a slight increase of contact time may facilitate binding.Place the tube in a Magnetic Separator (see accessories) or use a magnet to remove the supernatant and discard it.
  4. Follow this by washing the magnetic beads by adding 500uL of washing buffer (Washing buffer: 50 mM NaH2P04 , 300 mM NaCI, 20 mM imidazole, pH 8.0) and mix it using a vortex. Place the tube again in the magnetic separator and discard the supernatant. Repeat the previous step two more times.

    5. Elution of the Pure Protein

  5. Add 100 μl elution buffer (50 mM NaH2P04. 300 mM NaCI, 500 mM imidazole pH 8.0 )to the magnetic beads. Mix thoroughly for 10 min and place the tube in the magnetic separator and collect the elution fraction and store on ice. Repeat the elution step 2x or more and collect each fraction in a separate tube and determine the protein concentration of each fraction.
  1. Centrifuge E. coli cell suspension for 5 min at 14,000 g (4 °C) to collect the cells
  2. Discard the entire supernatant
  3. Resuspend the cells in ice-cold Cell Fractioning Buffer 1. The resulting volume should be 1/4 of the former suspension volume
  4. Incubate for 20 min on ice. Invert the suspension at regular intervals to counteract sedimentation
  5. Centrifuge the cell suspension for 15 min at 14,000 g (4 °C)
  6. Discard the entire supernatant
  7. Resuspend the cells in ice-cold Cell Fractioning Buffer 2. The resulting volume should be 1/4 of the former suspension volume
  8. Incubate for 10 up to 20 min on ice under regular invertion
  9. Centrifuge the cell suspension for 15 min at 14,000 g (4 °C)
  10. Save the supernatant, which contains the periplasmatic proteins and membrane proteins

Lysis Protocol

  1. A five ml culture of the bacterial strain containing the recombinant protein should be grown under the appropriate conditions for expression.
  2. [Save 200uL of the original overnight culture for SDS-PAGE: spin 200uL for 2 minutes. Remove supernatant and resuspend pellet in 100uL of 1x Loading Dye + DTT. Heat this sample tube to 95–100°C for 3–5 minutes]
  3. Use 1.5 ml of the bacterial culture and centrifuge the cells at full speed for 2 minutes.
  4. Remove the spent medium and add an additional 1.5mL of the bacterial culture.
  5. Centrifuge for 2 minutes.
  6. Remove the supernatant and resuspend the cell pellet in 0.4 ml of CelLytic B
  7. Briefly vortex the solution to resuspend the cell pellet and mix for 5-10 minutes to ensure full extraction of the soluble proteins.
  8. Centrifuge the cell lysate at full speed for 5 minutes to pellet any insoluble material.
  9. Carefully remove the soluble protein fraction (supernatant) from the cell debris to a clean microfuge tube. Save the pellet for the inclusion body protocol below.
  10. Prepare 30uL of supernatant for SDS-PAGE (following instructions for Sample Preparation below)

Inclusion body Protocol

  1. Inclusion bodies are the pellet obtained from the step 9 of the lysis protocol.
  2. Add 200 ul of CelLytic IB to the pellet.
  3. Vortex this suspension for 1 minute.
  4. Shake the suspension for 30 minutes at room temperature.
  5. Centrifuge the suspension at 13,000rpm for 15 minutes at room temperature to remove cell debris.
  6. After centrifugation, carefully remove the supernatant containing the soluble protein to a clean microfuge tube. This contains proteins extracted from the inclusion bodies. Discard the pellet.
  7. Precipitate the proteins in the supernatent via TCA precipitation.

TCA precipitation protocol

  1. Add 1 volume 100% (w/v) TCA stock to 4 volumes of protein sample. (i.e., for 200uL of supernatant, add 50uL of TCA).
  2. Incubate 10 min at 4 deg C
  3. Spin at 13000rpm for 5 min
  4. Remove and discard supernatant, leaving protein pellet intact. Pellet formed should be whitish
  5. and fluffy.
  6. Resuspend pellet with 200uL cold acetone.
  7. Spin at 13000rpm for 5 min and remove acetone.
  8. Repeat steps 5-6
  9. Dry pellet (evaporate acetone) by placing tube in 95deg C heat block for 5-10 min.
  10. To prepare pellet for SDS-PAGE, resuspend pellet in 100uL of 1x Loading Dye + DTT. Heat this sample tube to 95–100°C for 3–5 minutes

Sample Preparation

  1. Prepare fresh 3X Reducing Blue Loading Buffer by adding 1/10 volume 30X Reducing Agent to 1 volume of 3X Blue Loading Buffer.
  2. Prepare samples by adding 1/2 volume of 3X Reducing Blue Loading Buffer (from step 1).
  3. Heat samples to 95–100°C for 3–5 minutes.
  4. After a quick microcentrifuge spin, load directly on to a gel. To ensure uniform mobility, load an equal volume of 1X Reducing Blue Loading Buffer into any unused wells.

Make samples according to this chart:

SDS PAGE SAMPLES 30uL sample Variable Sample
Soluble Protein 19uL =X
30x DTT 1 =X/10
3x Loading Buffer 10uL
 =(X+X/10)/2