EXPERIMENTS

Double restriction enzyme digestion for pUC-IDT-qSCR plasmid

  1. Prepare the following materials:
    2. 10 μL plasmid DNA (0.6 mL) microcentrifuge tube x2
    A bottle of 10x restriction buffer sterile distilled water
    BamH1 reverse primer Nde1 forward primer
  2. Thaw all solutions and buffers needed for approximately 10 minutes. Homogenize the contents by gently flicking the tube. If possible, collect all liquid at the bottom of the tube by spinning briefly in the microcentrifuge at maximum speed. Place tubes on ice.
  3. Label a sterile 0.6 mL microcentrifuge tube with an identifying label for both forward and reverse RE you're doing.
  4. Pipette the following into the tube in the order listed by order. It adds up to a total volume of 20 μL:
    • 7 μL sterile distilled water
    • 10 μL plasmid DNA [*]
    • 2 μL 10x restriction buffer
    • 0.5 μL BamH1 reverse primer
    • 0.5 μL EcoR1 forward primer
  5. Mix gently by pipetting up and down a few times. You can bring all the liquid to the bottom of the tube by spinning it briefly in the microcentrifuge at maximum speed.
  6. Incubate at 37 °C in a water bath or thermocycler for 1 hour.
  7. Incubate at 80 °C for 20 minutes. This will denature the restriction enzymes.
  8. Store the restriction digests in -20 °C freezer

Double restriction enzyme digestion for PA1897_EGFP plasmid

  1. Prepare the following materials:
    2. 10 μL plasmid DNA (0.6 mL) microcentrifuge tube x2
    A bottle of 10x restriction buffer sterile distilled water
    BamH1 reverse primer Nde1 forward primer
  2. Thaw all solutions and buffers needed for approximately 10 minutes. Homogenize the contents by gently flicking the tube. If possible, collect all liquid at the bottom of the tube by spinning briefly in the microcentrifuge at maximum speed. Place tubes on ice.
  3. Label a sterile 0.6 mL microcentrifuge tube with an identifying label for both forward and reverse RE you're doing.
  4. Pipette the following into the tube in the order listed by order. It adds up to a total volume of 20 μL:
    • 7 μL sterile distilled water
    • 10 μL plasmid DNA [*]
    • 2 μL 10x restriction buffer>
    • 0.5 μL BamH1 reverse primer
    • 0.5 μL Nde1 forward primer>
  5. Mix gently by pipetting up and down a few times. You can bring all the liquid to the bottom of the tube by spinning it briefly in the microcentrifuge at maximum speed.
  6. Incubate at 37 °C in a water bath or thermocycler for 1 hour.
  7. Incubate at 80 °C for 20 minutes. This will denature the restriction enzymes.
  8. Store the restriction digests in -20 °C freezer

Double restriction enzyme digestion for PA1897_EGFP plasmid

  1. Prepare the following materials:
    2. 10 μL plasmid DNA (0.6 mL) microcentrifuge tube x2
    A bottle of 10x restriction buffer sterile distilled water
    BamH1 reverse primer Nde1 forward primer
  2. Thaw all solutions and buffers needed for approximately 10 minutes. Homogenize the contents by gently flicking the tube. If possible, collect all liquid at the bottom of the tube by spinning briefly in the microcentrifuge at maximum speed. Place tubes on ice.
  3. Label a sterile 0.6 mL microcentrifuge tube with an identifying label for both forward and reverse RE you're doing.
  4. Pipette the following into the tube in the order listed by order. It adds up to a total volume of 20 μL:
    • 7 μL sterile distilled water
    • 10 μL plasmid DNA [*]
    • 2 μL 10x restriction buffer
    • 0.5 μL BamH1 reverse primer
    • 0.5 μL Nde1 forward primer
  5. Mix gently by pipetting up and down a few times. You can bring all the liquid to the bottom of the tube by spinning it briefly in the microcentrifuge at maximum speed.
  6. Incubate at 37 °C in a water bath or thermocycler for 1 hour.
  7. Incubate at 80 °C for 20 minutes. This will denature the restriction enzymes.
  8. Store the restriction digests in -20 °C freezer

Single restriction Enzyme digestion for pUC-IDT-qSCR plasmid

  1. Prepare the following materials:
    2. 10 μL plasmid DNA (0.6 mL) microcentrifuge tube x2
    A bottle of 10x restriction buffer sterile distilled water
    BamH1 reverse primer
  2. Thaw all solutions and buffers needed for approximately 10 minutes. Homogenize the contents by gently flicking the tube. If possible, collect all liquid at the bottom of the tube by spinning briefly in the microcentrifuge at maximum speed. Place tubes on ice.
  3. Label a sterile 0.6 mL microcentrifuge tube with an identifying label for the reverse RE you're doing.
  4. Pipette the following into the tube in the order listed by order. It adds up to a total volume of 20 μL:
    • 7 μL sterile distilled water
    • 10 μL plasmid DNA [*]
    • 2 μL 10x restriction buffer
    • 1 μL BamH1 reverse primer
  5. Mix gently by pipetting up and down a few times. You can bring all the liquid to the bottom of the tube by spinning it briefly in the microcentrifuge at maximum speed.
  6. Incubate at 37 °C in a water bath or thermocycler for 1 hour.
  7. Incubate at 80 °C for 20 minutes. This will denature the restriction enzymes.
  8. Store the restriction digests in -20 °C freezer.

Single restriction Enzyme digestion for pET_IDT-qSCR plasmid

  1. Prepare the following materials:
    2. 10 μL plasmid DNA (0.6 mL) microcentrifuge tube x2
    A bottle of 10x restriction buffer sterile distilled water
    BamH1 reverse primer
  2. Thaw all solutions and buffers needed for approximately 10 minutes. Homogenize the contents by gently flicking the tube. If possible, collect all liquid at the bottom of the tube by spinning briefly in the microcentrifuge at maximum speed. Place tubes on ice.
  3. Label a sterile 0.6 mL microcentrifuge tube with an identifying label for the reverse RE you're doing.
  4. Pipette the following into the tube in the order listed by order. It adds up to a total volume of 20 μL:
    • 7 μL sterile distilled water
    • 10 μL plasmid DNA [*]
    • 2 μL 10x restriction buffer
    • 1 μL BamH1 reverse primer
  5. Mix gently by pipetting up and down a few times. You can bring all the liquid to the bottom of the tube by spinning it briefly in the microcentrifuge at maximum speed.
  6. Incubate at 37 °C in a water bath or thermocycler for 1 hour.
  7. Incubate at 80 °C for 20 minutes. This will denature the restriction enzymes.
  8. Store the restriction digests in -20 °C freezer

Single restriction enzyme digestion for PA1897_EGFP plasmid

  1. 1.Prepare the following materials:
    2. 10 μL plasmid DNA (0.6 mL) microcentrifuge tube x2
    A bottle of 10x restriction buffer sterile distilled water
    BamH1 reverse primer
  2. Thaw all solutions and buffers needed for approximately 10 minutes. Homogenize the contents by gently flicking the tube. If possible, collect all liquid at the bottom of the tube by spinning briefly in the microcentrifuge at maximum speed. Place tubes on ice.
  3. Label a sterile 0.6 mL microcentrifuge tube with an identifying label for the reverse RE you're doing.
  4. Pipette the following into the tube in the order listed by order. It adds up to a total volume of 20 μL:
    • 7 μL sterile distilled water
    • 10 μL plasmid DNA [*]
    • 2 μL 10x restriction buffer
    • 1 μL BamH1 reverse primer
  5. Mix gently by pipetting up and down a few times. You can bring all the liquid to the bottom of the tube by spinning it briefly in the microcentrifuge at maximum speed.
  6. Incubate at 37 °C in a water bath or thermocycler for 1 hour.
  7. Incubate at 80 °C for 20 minutes. This will denature the restriction enzymes.
  8. Store the restriction digests in -20 °C freezer.

Ligation

  1. Prepare the following materials:
    2. Insert restriction digest mix Vector restriction digest mix
    (0.6 mL) sterile microcentrifuge tube sterile distilled water
    10x ligation buffer
  2. Thaw all solutions and buffers needed. Homogenize the contents by gently flicking the tube. If possible, collect all liquid at the bottom of the tube by spinning briefly in the microcentrifuge at maximum speed. Place tubes on ice.
  3. Calculate the volume of insert RE mix based on the concentration of it by formula:

    4. M1V1 = M2V2


  4. Label a sterile 0.6 mL microcentrifuge tube with an identifying label for the particular ligation reaction you're doing. Make sure that you use a permanent marker and the tube fits inside the heating block of your thermocycler.
  5. Pipette the following into the tube in the order listed. It adds up to a total volume of 10 μL.
    • 3 μL sterile distilled water
    • 3 μL of insert restriction digest
    • 2 μL of vector restriction digest
    • 1 μL 10x ligation buffer
    • 1 μL T4 DNA Ligase
  6. Mix gently by pipetting up and down a few times. You can bring all the liquid to the bottom of the tube by spinning it briefly in the microcentrifuge at maximum speed.
  7. Incubate at room temperature for about 1 hour.
  8. Incubate at 80 °C for 20 minutes. This will denature the ligase, which otherwise may interfere with transformation.
  9. Store the ligation mix in a -20 °C freezer, or proceed immediately with transformation.

Transformation

  1. Prepare the following materials:
    2. control plasmid DNA DNA ligation mix
    SOC medium sterile distilled water
    100 μL aliquots of E. coli DH5ɑ competent cells LB agar plates (with desired antibiotic)
  2. Obtain a microcentrifuge tube containing 100 μL competent cells. These can be either freshly prepared or taken out of the -80 °C freezer. COMPETENT CELLS MUST BE THAWED AND KEPT ON ICE. Thawing on ice can take up to 10 minutes.
  3. Label the tube.
  4. Using a sterile pipette tip, add 25 μL ligation mix to the competent cells. Mix gently by flicking the tube. The rest of the ligation mix can be stored in a -20 °C freezer.
  5. Incubate on ice for 30 minutes.
  6. Incubate at 42 °C for 60 seconds.
  7. Place immediately on ice for 5 minutes.
  8. Add 200 μL SOC medium and mix gently by tapping the tube.
  9. Incubate at 37 °C for 2 hours.
  10. Warm up the LB agar plates to room temperature and label them. Make sure the plates contain the antibiotic that will select for your desired ligation product.
  11. Plate out 200 μL and 150 μL of the transformed cells onto two separate plates. Try to spread the cells evenly on the surface of the agar.
  12. Incubate plates at 37 °C overnight 16 hours. Make sure plates are incubated with a gar layer on top (and lid on the bottom).
  13. Plates with colonies can be stored at 4 °C (fridge) and can be used for at least a couple of weeks. Make sure plates are sealed with parafilm, labeled and oriented correctly (agar on top) while stored in the fridge.

Co-Transformation

  1. Prepare the following materials:
    2. control plasmid DNA DNA ligation mix pET_21a (+) _qSCR and PA1897_EGFP_mutant
    SOC medium sterile distilled water
    100 μL aliquots of E. coli DH5ɑ competent cells LB agar plates (with desired antibiotic)
  2. Obtain a microcentrifuge tube containing 100 μL competent cells. These can be either freshly prepared or taken out of the -80 °C freezer. COMPETENT CELLS MUST BE THAWED AND KEPT ON ICE. Thawing on ice can take up to 10 minutes.
  3. Label the tube.
  4. Using a sterile pipette tip, add 25 μL ligation mix pET_21a (+) _qSCR to the competent cells. Mix gently by flicking the tube. The rest of the ligation mix can be stored in a -20 °C freezer. Repeat this same step with PA1897_EGFP_mutant.
  5. Incubate on ice for 30 minutes.
  6. Incubate at 42 °C for 60 seconds.
  7. Place immediately on ice for 5 minutes.
  8. Add 200 μL SOC medium and mix gently by tapping the tube.
  9. Incubate at 37 °C for 2 hours.
  10. Warm up the LB agar plates to room temperature and label them. Make sure the plates contain double selective markers of Kan and Amp.
  11. Plate out 200 μL and 150 μL of the transformed cells onto two separate plates. Try to spread the cells evenly on the surface of the agar.
  12. Incubate plates at 37 °C overnight 16 hours. Make sure plates are incubated with a gar layer on top (and lid on the bottom).
  13. Plates with colonies can be stored at 4 °C (fridge) and can be used for at least a couple of weeks. Make sure plates are sealed with parafilm, labeled and oriented correctly (agar on top) while stored in the fridge.

Mini prep Qiagen kit

  1. Prepare the following materials:
    2. All Qiagen buffers Competent cells with desired plasmid
    1.5 μL microcentrifuge tubes x4 spinning column & collection tubes x2
  2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a micro- centrifuge tube.
  3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 6 times.
  4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 6 times.
  5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
  6. Apply the supernatants from step 4 to the QIAprep spin column by pipetting.
  7. Centrifuge for 60 s. Discard the flow-through.
  8. Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 60 s. Discard the flow-through.
  9. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 60 s.
  10. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
  11. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.
  12. Centrifuge for another 1 min, collect DNA samples.

Q5® Site-Directed Mutagenesis Kit

  1. Prepare the following materials:
    2. Q5 Hot Start High-fidelity 2X Master Mix 10 μL Forward Primer
    10 μL Reverse Primer Template DNA (1-25 ng/ μL)
    100 μL aliquots of competent cells sterile distilled water
  2. Exponential Amplification:
    Set up the PCR machine as following:
    3. Step temperature Time
    Initial denaturation 98°C 30s
    25 cycles 98 °C (denaturation) →
    67°C (annealing) →
    72 °C (extension)     		10s
    30s
    20s
    Final extension 72°C 2 mins
    Hold 4°C Forever
  3. KLD reaction
    Prepare the following materials:
    4. Volume Final concentration
    PCR Product 1 μL N/A
    2X KLD reaction buffer 5 μL 1X
    10X KLD Enzyme Mix 1 μL 1X
    Nuclease free water 3 μL N/A

    Incubate for 5 minutes at R.T.

  4. Transformation
    • Add 5 μL of KLD mix to 50 μL of competent cells.
    • Incubate on ice for 30 minutes
    • Heat shock at 42 °C for 30 seconds
    • Incubate on ice for 5 minutes
    • Add 950 μL SOC, gently shake at 37°C for 1 hour
    • Spread 100 μL onto appropriate selection plate, incubate ON at 37°C

Running agarose gel electrophoresis

  1. Prepare the following materials:
    2. 10x TAE buffer agrose
    DNA stain (e.g. Sybr Safe) DNA loading buffer 6x
    DNA restriction digest DNA ladder
    apparatus for agarose gel electrophoresis (gel tray, comb, tank, power supply with connectors)
  2. Prepare 100 mL 1 x TAE buffer by adding 10 mL 10 x TAE buffer to a cylinder containing 90 mL distilled water. Mix well with a clean stir rod. This volume should be good for most gel cassettes.
  3. Pour the 1 x TAE buffer into an Erlenmeyer flask. There should be enough empty space left in the flask to keep the agarose from boiling over during microwaving.
  4. Weigh out 1 g agarose powder and add it to the 1 x TAE buffer in the flask.
  5. Place the flask inside the microwave and set at maximum heat for 1 minute. Pause the microwave every 15 seconds to give the flask a couple of swirls. This will help in dissolving the agarose and in preventing the solution from boiling over. Repeat as needed, until all agarose has dissolved.
  6. Wait for the agarose solution to cool down but do not wait too long as it may solidify inside the flask. Assemble the gel cassette by sealing off the gel tray with tape. Place the comb in the proper location.
  7. Add 4 μL DNA stain (e.g. Sybr Safe) to the cooled agarose. Mix by swirling. Avoid forming air bubbles in the agarose.
  8. Pour the agarose solution carefully into the cassette. Avoid forming and trapping air bubbles.
  9. Wait for agarose to solidify.
  10. Prepare the DNA sample by mixing the following in a clean microcentrifuge tube to a total volume of 12 μL (see Note 1). Always include the dna ladder along with your samples.
    • 2 μL 6 x Loading Buffer
    • 4 μL DNA restriction digest
    • 6 μL distilled water
  11. Peel the tape off the gel and remove the comb carefully.
  12. Place the tray with the agarose gel in the tank for electrophoresis. Make sure the wells are located at the negative end of the electrical field.
  13. Fill the tank with a 1 x TAE buffer. Cover the gel with a layer of buffer 3-5 mm thick. If air bubbles get trapped inside the wells, displace them carefully using a clean pipette tip.
  14. Load your DNA sample(s) and the DNA ladder into the wells. Make sure to keep a record of which sample went into which well.
  15. Hook up the gel tank to the power supply. The wells with DNA must be on the negative (black) side!
  16. Turn on the power and run the gel at 100 V for at least 1 hour. The gel must stay covered throughout the run.
  17. Switch off the power when the dye has traveled at least 3⁄4 of the gel length and has neared the positive end of the gel.
  18. Unplug the gel tank. Take the gel out of the tank and view the DNA bands using a blue-light gel machine. Take a picture of the gel and record the results.

PCR

  1. Prepare the following materials (reverse and forward depending on plasmids):
    2. sterile distilled water sterile microcentrifuge tubes
    10 x PCR buffer 10 mM DNTPs mix
    10 μM forward primer 10 μM reverse primer
    50 mM MgCl2 solution Taq Polymerase
    DNA template
  2. Prepare the following master mix for each PCR tubes:
    3. Ingredient Volume
    sterile distilled water 35 μL
    10 x PCR buffer 5 μL
    10 mM dNTPs mix 1 μL
    10 μM forward prime 1 μL
    10 μM reverse prime 1 μL
    50 mM MgCl2 1.5 μL
    Taq Polymerase 0.5 μL
  3. Pipet 5 μL DNA template into each PCR tube. The total amount of template DNA can vary between 1 pg and 1μg depending on the source (very little is needed if the template is pure plasmid DNA; significantly more will be needed if the template you use is chromosomal/genomic DNA)
  4. Add 45 μL of the master mix to each of the PCR tubes. Mix gently by tapping the tubes or by pipetting up and down a few times. If needed, spin down briefly at maximum speed in the microcentrifuge to collect all liquid at the bottom of tubes.
  5. Top the PCR reactions with mineral oil if your PCR machine doesn't have a heated lid.
  6. Transfer the tubes to PCR machine and start the program (you'll need to program your machine first):
    7. Step temperature Time
    Initial denaturation 98°C 30s
    25 cycles 98 °C (denaturation) →
    67°C (annealing) →
    72 °C (extension)     		10s
    30s
    20s
    Final extension 72°C 2 mins
    Hold 4°C Forever

Making glycorel stocks

  1. Prepare the following materials:
    2. sterile glycorel solution sterile microcentrifuge tubes
    bacterial culture of interest -80℃ freezer
  2. Obtain a couple of sterile microcentrifuge tubes and label them.
  3. Use a sterile pipet tip to transfer 0.5 mL of the bacterial culture of interest into each of the tubes.
  4. Use a sterile pipet tip to add 0.5 mL of an 80 % glycerol solution to each tube. Mix by pipetting up and down.
  5. Place in an -80 °C freezer for long-term storage.

References

*: The volume of plasmid DNA diluted with nuclease free water depends on the concentration we get from Nanodrop spectrometer. We use the following formula to calculate the volume of plasmid.


M1V1 = M2V2

M1 stand for the molar concentration of initial plasmid, V1 stand for the volume of initial plasmid. M2 stand for the molar concentration of working plasmid, V2 stand for the volume of working plasmid. We start by setting our working plasmid concentration as 20 ngμL-1 , working volume as 5 μL, inputting the concentration of initial plasmid that we get from Nanodrop spectrometer and calculate can give us the volume to be added for initial plasmid. The rest of the few μL we will add sterile distilled water to dilute it to the working concentration.