Team:SASTRA Thanjavur/Experiments

PROTOCOLS
Index
      
High Efficiency Transformation Protocol by NEB - modified (NEB#29871)

Materials:

  1. NEB#C2987I
  2. LB medium
  3. Crushed Ice
  4. Microcentrifuge tubes
  5. Pipettes and tips


Procedure:

  1. Thaw a tube of NEB 5-alpha competent E.coli cells on ice. Gently mix the tube.
  2. Transfer 50 µL of cells into an microcentrifuge on ice.
  3. Add 3 µL of Plasmid DNA to the cell mixture. Carefully flick the tube to mix the cells and the DNA. (DO NOT VORTEX)
  4. Allow the solution to remain undisturbed on ice for 30 minutes.
  5. Heat shock at exactly 42 ℃ for 30 seconds. Do not mix the tube.
  6. Place the tube on ice for 5 minutes.
  7. Pipette 950 µL of room temperature LB into the mixture.
  8. Incubate the mixture for 60 minutes at 37 ℃. Shake the tube at 250 rpm.
  9. Spin the cell suspension at a low rpm and remove the supernatant. Resuspend the cell pellet in LB medium and plate the cell suspension on LB agar with a concentration of 35 µg/mL Chloramphenicol.
  10. Incubate the plates at 37 ℃ for 24 hours to obtain colonies of the transformed cells.


      
DNA Resuspension Protocol (For BioBrick Bba_K608011)

Materials:

  1. DNA distribution plates
  2. Distilled water
  3. Chloramphenicol
  4. Pipettes and tips


Procedure:

  1. Punch a hole in the well containing the desired biobrick, through the foil cover.
  2. Pipette 10 µL of distilled water into the well.
  3. Mix the contents in the well gently, with a pipette.
  4. Leave the plate undisturbed for 5 minutes.
  5. Resuspension of the DNA is indicated by the presence of a red color due to the crescent dye.
  6. Use the resuspended DNA to transform the competent cells using the high efficiency transformation protocol


      
PCR Protocol

To amplify the toehold construct for cell free reactions (miRNA 21 and 20a)

  1. Add TE buffer to resuspend the gblock to your final desired concentration
  2. Vortex it briefly to suspend it
  3. It can be stored at -80 ℃ upto 2 years
  4. Freeze thaw the master mix before usage
  5. Dilute the gblocks to the required concentration using nuclease free TE buffer
  6. Keep all reaction components on ice and add them to a PCR tube  and transfer the reactions immediately to the thermocycler
  7. The thermocycler should be preheated to the denaturation temperature and the components must be well mixed before transferring
  8. Allow the thermocycler to run to the set cycle parameters.
  9. Run the PCR product on a 1.5% agarose gel and check the amplicon size
  10. The product can be purified by using a purification kit and sequenced to confirm the amplification of gblocks

COMPONENTS

25 µL reaction

15 µL reaction

Q5 High fidelity 2X master mix (NEB kit)

12.5 µL

7.5 µL

10 uM FP (VF2)

1.25 µL

0.75 µL

10 uM RP (VR)

1.25 µL

0.75 µL

Template DNA (1ng)

1 µL - 3µL

1-3 µL

Nuclease free water

6-9 µL

6-9 µL

GRADIENT PCR (Temperature set up and no. of cycles)

98℃- denaturation (initial)

30 seconds

98℃

10 s

62/63℃ - annealing temperature

20s

72℃- extension

30s

72℃ -final extension

3 minutes

4℃

∞
      
Characterization of Biobrick Bba_K608011 pH experiment Protocol

Materials:

  1. LB Broth
  2. Centrifuge Tubes
  3. Chloramphenicol
  4. 1M NaOH
  5. 35% HCl
  6. Transformed E.Coli, containing Biobrick Bba_K608011
  7. pH meter, fluorescence spectrophotometer
  8. Pipettes and tips


Procedure:

  1. Prepare 300 mL of LB broth in distilled water.
  2. Separate the LB broth into 6 different 50 mL centrifuge tubes, each containing 40 mL of the prepared broth.
  3. Prepare 1N NaOH and 35% HCl. Adjust the pH of the broth in 5 of the tubes to the desired values of 5,6,7.2 (pH of standard LB broth), 8 and 9.
  4. Keep a separate tube with 40 mL of the broth (with unaltered pH) to use as a control.
  5. Autoclave the Tarson tubes.
  6. Add Chloramphenicol to all the Tarson tubes to obtain a final antibiotic concentration of 20 µg/mL. This was done by initially creating a stock of 40 mg/mL.
  7. Leave the LB Broth undisturbed overnight to check for contamination.
  8. Separate the 40 mL into 4 different Tarson tubes containing 10mL each- 3 of them to perform the experiment in triplicates and 1 tube, kept aside with LB for dilution to adjust Optical Density before fluorescence measurement.
  9. Inoculate the triplicate tubes at pH 5, 6, 7.2, 8 and 9 with the transformed E.Coli cells containing the biobrick
  10. At this stage, each Tarson tube at pH values of 5, 6, 7.2, 8 and 9 contain:
    • 10 mL LB broth at the particular pH
    • Chloramphenicol- concentration of 20 µg/mL
    • Inoculated cells
  11. Place the tubes in the orbital shaker at 37º Celsius for 48 hours until turbidity is visible.  Measure the OD (595nm) of the cultures grown under different pH values and normalize the OD to a constant value by diluting the samples with plain LB medium kept aside at that pH. (We used an OD value of 0.35, which was the OD obtained at pH 8).
  12. Measure the fluorescence intensity of the cell solutions (triplicates), keeping uninoculated LB broth as the control. Excitation at 485 nm and Emission at 520 nm.


      
Characterization of Biobrick Bba_K3154001 miR-21 2nd generation toehold switch

Materials:

  1. 96 - U bottom fluorescent well plate
  2. Toehold DNA
  3. miR & anti-miR
  4. NEB (#E6800 Pure Synthesis Kit)
  5. Micropipette 
  6. Fluorescent plate reader


Concentration study:

  1. Freeze thaw miR, anti-miR in ice for 5 mins.
  2. Quick spin miR and anti-miR @1000rpm (<1min)
  3. Resuspend the miR & anti-miR with T10E0.1 to obtain 100 µM stock solution

    miR type

    Volume of TE buffer added (mL)

    Anti-miR type

    Volume of TE buffer added (mL)

    21

    3.144

    21

    2.569

    20a

    2.896

    20a

    3.116

    200a

    2.401

    200a

    2.863

    29a

    2.532

    29a

    2.592
  4. Dilute the respective miR and anti-miR to prepare the working stock of 100 nM
  5. Mix 100nM of miR & anti-miR solution of required volume (50 µL each)
  6. Incubate for 2hrs at room temperature to facilitate hybridization of miR and anti-miR 
  7. Freeze thaw:
    1. Solution A & Solution B from NEB #E6800 cell free protein expression kit
    2. Toehold DNA obtained after PCR & purification with required DNA concentration (125 ng/µL)

  8. Prepare the master mix containing solution A (10 µL) and solution B (7.5 µL)
  9. Prepare the varying concentrations (100pM, 1nM, 10nM, 100nM) of the miR – anti-miR (MA) complex
  10. Mix the following contents:
    1. Master Mix = 17.5 µL
    2. Toehold DNA = 1 µL (21); 3 µL (33ng/µL)-4 µL (26ng/µL) (20a)
    3. MA = 2 µL
    4. Nuclease free water = X µL
      5. Total = 30 µL

  11. For specificity study, perform the same steps but add MA at a fixed concentration (100 nM in our case) that does not correspond to the respective toehold DNA. Example – add MA of 29a to Toehold DNA for miR 21. 
  12. Incubate the reaction systems at 37 °C and perform fluorescence intensity measurements at 2nd and 4th hr with the following settings:
    1. Excitation Wavelength = 395 nm 
    2. Emission Wavelength = 520 nm
    3. System Manual Gain = 100




References
  1. https://international.neb.com/protocols/0001/01/01/high-efficiency-transformation-protocol-c2987
  2. http://parts.igem.org/Help:2018_DNA_Distribution
  3. https://international.neb.com/protocols/0001/01/01/taq-dna-polymerase-with-standard-taq-buffer-m0273
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