Experiments

All Protocols in One Place

Experiments

Cloning Protocols
Rolling Circle Amplification
Rolling Circle Transcription
Proof of Concept

Cloning Protocols

Restriction Digest

Reagent Amount
Restriction Enzyme 1 1 µL
Restriction Enzyme 2 1 µL
10x Cutsmart Buffer 5 µL
Insert or Vector DNA 1 µg
Nuclease Free Water To 50 µL
  1. Shave ice in a styrofoam container.
  2. Label PCR tubes.
  3. Add all reagents to the tube.
  4. Incubate for 30 minutes at 37°C. If both enzymes are Time-Saver Qualified, only incubate for 5-15 minutes.
  5. Incubate for 20 minutes at 65°C.

Ligation

Reagent Amount
T4 Ligase Buffer 2 µL
T4 DNA Ligase 1 µL
Vector DNA 50 ng
Insert DNA 150 ng
Nuclease Free Water To 20 µL
  1. Shave ice in a styrofoam container.
  2. Label PCR tubes.
  3. Add all reagents to the tube.
  4. Incubate for 10 min at room temperature.
  5. Incubate for 10 min at 65°C.

Transformation

  1. Label tubes with initials, date, and what is being transformed.
  2. Place all tubes on ice.
  3. Put SOC/LB and antibiotic plates in the incubator to warm at 37°C.
  4. Pipette 1 µL of the desired plasmid(s) and 1 µL of PUC19 into the corresponding labeled transformation tubes.
  5. Thaw competent cells on ice for 10 minutes or just until the cells inside are moving when you flick the tube.
  6. Pipette 50 µL of competent cells into each labeled transformation tube on ice.
  7. Incubate tubes on ice for 30 minutes.
  8. Heat shock tubes at 42°C for 45 seconds.
  9. Place on ice for 2 minutes.
  10. Add 950 µL of SOC/LB media into each tube with cells.
  11. Place tubes shaking in the incubator for 60 minutes at 37°C.
  12. Pipette 120-150 µL of cells with SOC/LB media onto warm plates with the correct antibiotics.
  13. Place antibiotic plates in the incubator at 37°C for 24 hours to let cells grow.

Inoculation for Culture

  1. Prepare liquid LB.
  2. Add LB to a tube or flask.
  3. Add 1 µL of antibiotic for every 1 mL of LB (typically use 5 µL : 5 mL).
  4. Antibiotics should correspond to the cell’s antibiotic resistance gene.
  5. Using a sterile pipette tip, pick up a single colony from your LB agar plate.
  6. Drop the tip into liquid LB and antibiotic, then swirl to release cells.
  7. Cover culture with sterile aluminum foil or cap that is not airtight.
  8. Incubate culture at 37℃ for 12-18 hours in a shaking incubator.
  9. After incubation, check for growth.
  10. For long-term storage, continue with creating a glycerol stock.

Miniprep

  1. Grow 1-5 mL culture overnight in a 10 mL - 20 mL culture tube.
  2. Centrifuge at 2500 xg for 10 minutes at room temperature. Decant or aspirate and discard culture media.
  3. Add 250 µL of solution 1 mixed with RNase. Vortex to mix, then transfer suspension into a new 1.5 mL microcentrifuge tube.
  4. Add 250 µL of solution 2. Invert until there is a clear lysate.
  5. Add 350 µL to solution 3. Invert until white precipitate forms. Centrifuge at 13,000 xg for 10 minutes.
  6. Insert mini column into a 2 mL collection tube.
  7. Transfer the clear supernatant into a mini-column using a micropipette. Centrifuge at 13,000 xg for 60 seconds. Discard filtrate and reuse the collection tube.
  8. Add 500 µL of HBC Wash Buffer diluted in ethanol. Centrifuge at 13,000 xg for 60 seconds. Discard filtrate and reuse the collection tube.
  9. Add 700 µL of the DNA Wash Buffer diluted in ethanol. Centrifuge at 13,000 xg for 30 seconds. Discard filtrate, and reuse collection tube.
  10. Centrifuge empty mini-column at 13,0000 xg for 2 minutes to remove ethanol.
  11. Transfer mini-column to a nuclease-free 1.5 mL microcentrifuge tube.
  12. Add 50 µL of Elution Buffer. Let it sit at room temperature for 5 minutes. Centrifuge at 13,000 xg for 60 seconds.
  13. Store eluted DNA at -20℃.

Gel Electrophoresis

Making Gels

  1. Mix 0.5 g of agarose with 50 mL of 1x TAE buffer in a 100 - 250 mL Erlenmeyer flask.
  2. Heat up the solution until it turns clear.
  3. Cool solution to 65°C.
  4. Add 6 µL of SYBR™ Safe and gently swirl the flask to mix the solution.
  5. Pour the solution into a gel chamber and put in the gel comb.
  6. Wait for the gel to solidify before loading.

Running the Gel

  1. Once solidified, fix the gel’s position so that the wells of the gel are at the end of the chamber and DNA runs to red.
  2. Pour used 1x TAE buffer into the gel chamber evenly to completely cover the gel.
  3. On a piece of parafilm, mix 5 µL of DNA and 1 µL of purple loading dye using a micropipette.
  4. Add the mixed 6 µL solution into each well.
  5. Add 6 µL of a ladder into an empty well.
  6. Close the gel chamber and connect electrodes to the power supply.
  7. Set power supply with voltage and time.
  8. Turn on the power supply and make sure bubbles are rising on the sides of the gel chamber.
  9. Once gel electrophoresis is completed, put the gel under UV (ultraviolet) light to compare the bands of DNA to the bands of the ladder.

PCR

Reagent Amount
Q5 High Fidelity Master Mix 12.5 µL
10 μM diluted forward primer 1.25 µL
10 μM diluted reverse primer 1.25 µL
Template DNA 1 µL
Sterile Water To 25 µL
  1. Label PCR tubes.
  2. Add reagents to PCR tube.
  3. Set thermocycler with temperatures and times (annealing temperature and elongation time can vary depending on part).
  4. Run thermocycler.

Colony PCR

Reagent Amount
Q5 High Fidelity Master Mix 12.5 µL
10 μM diluted forward primer 1.25 µL
10 μM diluted reverse primer 1.25 µL
Diluted Colony 1 µL
Sterile Water To 25 µL
  1. Dilute colony from LB agar plate into 40 µL of water.
  2. Use 1 µL for colony PCR reaction.
  3. Label PCR tube.
  4. Add reagents to PCR tube.
  5. Run in Thermocycler.

Glycerol Stock

  1. Grow 1-5 mL culture overnight in a 10 mL - 20 mL culture tube.
  2. Centrifuge at 2500 xg for 10 minutes at room temperature. Decant or aspirate and discard culture media.
  3. Remove all but 500 µL of growth media.
  4. Add 500 µL of glycerol stock and resuspend.
  5. Transfer solution to 1.5 mL microcentrifuge tube and store in -80°C.

Rolling Circle Amplification (RCA)

SYBR™ Safe Experimentation

1. miRNA - DNA hybridization and Ligation Reaction

  1. Make sure all the PCR tubes are RNAse free. Label the PCR tubes.
  2. Label three PCR tubes A1. Label three PCR tubes A2. Label three PCR Tubes A3. Label three PCR Tubes A4. Label three PCR Tubes A5. Label three PCR Tubes A6. Label three PCR Tubes A7. Label three PCR Tubes A8. Label three PCR Tubes A9. Label three PCR Tubes A10. Pipette 1 µL of 10 x Ligation Buffer (from NEB) into all of the PCR tubes.
  3. Add 0.5 µL of 100 nM padlock probe. Add .1 µL of 1000 pM of the miRNA-1. Add 6.9 µL of water.
  4. Heat the solution at 65°C for 3 minutes, and slowly cool it to room temperature over 10 minutes through the use of a thermocycler.
  5. Then, add 20 U or 0.5 µL of RNase inhibitors.
  6. Add 1 µL of Splint R Ligase.
  7. Incubate the reaction for 2 hours at 37°C.
  8. Bring reaction up to 65°C for 10 minutes to terminate. The reaction was brought up based on gradient 63-68°C. In addition, to determine the ideal situations where the miRNA and the padlock do not detach, the reaction was brought up to the gradient temperatures for 10 minutes as well as 20 minutes to determine the ideal temperature.
    • Each of the A1- A5 tubes are on the gradient for 10 minutes. Each of the A6- A10 tubes are on the gradient for 20 minutes.

2. RCA Reaction (Reaction volume will be 25 µL so add 0.5 µL of water)

  1. Pipette 2.5 µL of 10x phi29 buffer (from NEB) into previous ligation reaction mixture.

  2. Add 12.5 units or 1.25 µL of phi29 DNA polymerase.

  3. Add 6.75 ul of 10 mM dNTPs.

  4. Incubate the reactions for 8 hours at 37°C.

  5. Bring up the reactions to 65°C for 10 minutes to inactivate the DNA polymerase.

3. Readout

  1. The readout reaction utilizes Sybr Safe; use a 5:1 ratio of reaction volume: SYBR™ Safe.

  2. Run all the reactions through the plate reader to obtain fluorescence values.

Gel Experimentation

1. miRNA - DNA hybridization and Ligation Reaction

  1. Pipette 1 μL of NEB 10 x Ligation Buffer into pcr tubes.
  2. Add 2.5 μL of 20 nM padlock probe . For miRNA, do a 1:10 dilution using a 1000 pM stock and add 1 μL of diluted miRNA into reaction.
  3. Heat the solution at 65°C for 3 minutes, and slowly cool it to room temperature over 10 minutes.
  4. Add 1 μL of Splint R Ligase and 0.5 μL of RNAse inhibitor into sample.
  5. Incubate the reaction for 2 hours at 37°C.
  6. Bring reaction up to 65°C for 20 minutes to terminate.

2. RCA Reaction

  1. Pipette 2.5 μL of NEB 10x phi29 buffer into previous ligation reaction mixture.
  2. Add 12.5 units or 1.25 μL of phi29 DNA polymerase. Add 1 μL of aluminum.
  3. Add 6.76 μL of 10 mM dNTPs.
  4. Incubate the reactions for 8 hours at 37°C.
  5. Bring the reactions up to 65°C for 10 minutes to inactivate the DNA polymerase.

3. Readout

  1. Add 10 μL of reaction + 2 μL of loading dye into 1% agarose gel with SYBR™ Safe.

Detection Limit Experimentation

The following concentrations of miRNA were used to determine the lower detection limit:

  1. Add 5 µL of 1000 pM of the miRNA-1.
    • Final Concentration: 204 pM
  2. Add 1 µL of 1000 pM of the miRNA-1.
    • Final Concentration: 40.8 pM
  3. Add 0.5 µL of 1000 pM of the miRNA-1.
    • Final Concentration: 20.4 pM
  4. Add 1 µL of 100 pM of the miRNA-1.
    • Final Concentration: 4.08 pM
  5. Add 0.8 µL of 100 pM of the miRNA-1
    • Final Concentration: 3.27 pM
  6. Add 0.5 µL of 100 pM of miRNA-1
    • Final Concentration: 2.04 pM
  7. Add 0.3 µL of 100 pM of miRNA-1.
    • Final Concentration: 1.22 pM
  8. Add 1 µL of 10 pM of miRNA-1.
    • Final Concentration: 0.408 pM
  9. Add 0.5 µL of 10 pM of miRNA-1.
    • Final Concentration: 0.204 pM
  10. Add 3 µL of 1 pM of miRNA-1.
    • Final Concentration: 0.122 pM
  11. Add 1 µL of 1 pM of miRNA-1.
    • Final Concentration: 0.0408 pM

Lettuce with Complement

  1. In nuclease-free PCR tubes add the following triplicates:
    Dye Control (Background)
    • 25 μL water
    • 2.5 μL phi29 DNA polymerase buffer
    • 1 μL SplintR Ligase buffer
    Lettuce Left + Lettuce Right + Dye
    • 19 μL RNase-free water
    • 1 μL 10X Splint R Ligase Buffer
    • 2.5 μL 10X phi29 Reaction Buffer
    • 3 μL Split Lettuce Left Sequence (100 uM stock)
    • 3 μL Split Lettuce Right Sequence (100 uM stock)
    Complement + Lettuce Left + Lettuce Right + Dye
    • 17.5 μL RNase-free water
    • 3 μL Split Lettuce Left Sequence (100 uM stock)
    • 3 μL Split Lettuce Right Sequence (100 uM stock)
    • 1.5 μL complement
    • 1 μL 10X Splint R Ligase Buffer
    • 2.5 μL 10X phi29 Reaction Buffer
  2. Centrifuge the reagents
  3. On the sides of the centrifuged PCR tubes, carefully add 1.5 μL of DFHBI-1T (100 uM stock)
  4. For controls without the dye, repeat steps 1-3 were repeated with 1.5 μL RNase-free water being added in place of the dye.
  5. Vortex and centrifuge reagents.
  6. Incubate the reaction at 70°C for 5 minutes, then cool to and incubate at 25°C for 1 hour.
  7. Read fluorescence on the plate reader. The emission spectra is 528 nm, while the excitation spectra is 480 nm.

Lettuce with RCP

  1. In nuclease-free PCR tubes add the following triplicates:
    Dye Control (Background)
    • 25 μL water
    • 2.5 μL phi29 DNA polymerase buffer
    • 1 μL SplintR Ligase buffer
    Lettuce Left + Lettuce Right + Dye
    • 19 μL RNase-free water
    • 1 μL 10X Splint R Ligase Buffer
    • 2.5 μL 10X phi29 Reaction Buffer
    • 3 μL Split Lettuce Left Sequence (100 uM stock)
    • 3 μL Split Lettuce Right Sequence (100 uM stock)
    Complement + Lettuce Left + Lettuce Right + Dye
    • 17.5 μL RNase-free water
    • 3 μL Split Lettuce Left Sequence (100 uM stock)
    • 3 μL Split Lettuce Right Sequence (100 uM stock)
    • 1.5 μL complement
    • 1 μL 10X Splint R Ligase Buffer
    • 2.5 μL 10X phi29 Reaction Buffer
  2. Centrifuge the reagents
  3. On the sides of the centrifuged PCR tubes, carefully add 1.5 μL of DFHBI-1T (100 uM stock)
    • For controls without the dye, repeat steps 1-3 were repeated with 1.5 μL RNase-free water being added in place of the dye.
  4. Vortex and centrifuge reagents.
  5. Incubate the reaction at 70°C for 5 minutes, then cool to and incubate at 25°C for 1 hour.
  6. Read fluorescence on the plate reader. The emission spectra is 528 nm, while the excitation spectra is 480 nm.

Linear DNA Probes with Complement

  1. Add the following to an amber microcentrifuge tube to make FAM-Probe Mastermix:
    FAM Dye Tagged Linear DNA Probe Mastermix
    • Volume of FAM Dye Tagged Linear DNA Probe: (# of reactions) x (1.6 µL FAM Dye Tagged Linear DNA Probe) x 1.1
    • Volume of TE: (# of reactions) x (29 µL TE) x 1.1
  2. Add the following to a PCR tube:
    Tris-EDTA Buffer (Background)
    • 34 µL TE Buffer
    FAM Dye Tagged Linear DNA Probe + TE
    • 1.6 µL BHQ-1 Quencher Tagged Probe
    • 32.4 µL TE Buffer
    BHQ-1 Quencher Tagged Linear DNA Probe + TE
    • 1.6 µL BHQ-1 Quencher Tagged Probe
    • 32.4 µL TE Buffer
    FAM Dye Tagged Linear DNA Probe + BHQ-1 Quencher Tagged Linear DNA Probe + Linear Probe Complement + TE
    • 29.4 µL Mastermix
    • 1.6 µL BHQ-1 Quencher Tagged Probe
    • 3 µL Linear Probe Complement
  3. Vortex several seconds and spin down tubes.
  4. Place tubes in thermocycler at 41°C for 1 minute.
  5. Place tubes in thermocycler 37°C for 1 minute.
  6. Pipette all 32 μL of solution into a 384 well plate to measure fluorescence at excitation wavelength of 480 nm and emission intensity at 518 nm using a plate reader.

Linear DNA Probes with RCP

  1. Add the following to an amber microcentrifuge tube to make FAM-Probe Mastermix:
    FAM Dye Tagged Linear DNA Probe Mastermix
    • Volume of FAM Dye Tagged Linear DNA Probe: (# of reactions) x (0.4 µL FAM Dye Tagged Linear DNA Probe) x 1.1
    • Volume of TE: (# of reactions) x (29 µL TE) x 1.1
    Tris-EDTA Buffer (Background)
    • 34 µL TE Buffer
    FAM Dye Tagged Linear DNA Probe + TE
    • 29.4 µL Mastermix
    • 4.6 µL TE Buffer
    BHQ-1 Quencher Tagged Linear DNA Probe + TE
    • 1.6 µL BHQ-1 Quencher Tagged Probe
    • 32.4 µL TE Buffer
    FAM Dye Tagged Linear DNA Probe + BHQ-1 Quencher Tagged Linear DNA Probe + Padlock Probe + miRNA
    • 29.4 µL Mastermix
    • 1.6 µL BHQ-1 Quencher Tagged Probe
    • 3 µL Padlock + miRNA Solution
    FAM Dye Tagged Linear DNA Probe + BHQ-1 Quencher Tagged Line0ar DNA Probe + Rolling Circle Product + TE
    • 29.4 µL Mastermix
    • 1.6 µL BHQ-1 Quencher Tagged Probe
    • 3 µL Linear Probe Rolling Circle Product
  2. Vortex several seconds and spin down tubes.
  3. Place tubes in thermocycler at 41°C for 1 minute.
  4. Place tubes in thermocycler 37°C for 1 minute.
  5. Pipette all 32 μL of solution into a 384 well plate to measure fluorescence at excitation wavelength of 480 nm and emission intensity at 518 nm using a plate reader.

Rolling Circle Transcription (RCT)

Overview of Steps

  1. Ligation Reaction
  2. RCT Reaction
  3. Aptamer Reaction

1. Ligation Reaction

  1. Pipette 6.5 μL of RNase-free water into RNase-free PCR tube
  2. Add 1 μL of 10x Ligation Buffer
  3. Add 0.5 μL of cDNA (padlock) stock and 1.0 μL of miR-1 stock (Stock: 1,000 pM diluted ten-fold)
  4. Incubate the mixture at 65°C for 3 minutes
  5. Cool the mixture to 25°C over 10 minutes
  6. Add 25U (1.0 μL) of Splint R ligase
  7. Incubate the mixture at 37°C for 2 hours
  8. Incubate the mixture at 65°C for 20 minutes to inactivate the Splint R ligase

2. RCT Reaction

  1. Pipette 16.3 μL of RNase-free water into ligation product
  2. Pipette 2 μL of 10x transcription buffer
  3. Add the following to the ligation product:
    • 40 U (0.8 μL) T7 RNA polymerase (from Stock: 50,000 units/mL)
    • 20 U (0.5 μL) RNase inhibitor (from Stock: 40,000 units/mL)
    • 0.4 μL NTPs (Stock: 25 mM)
  4. Incubate the mixture at 37°C for 8 hours
  5. Inactivate T7 RNA polymerase at 70°C for 10 minutes

3. Aptamer Reaction

  1. Directly add 40 μM of DFHBI-1T to the reaction mixture
  2. Read fluorescence values using a plate reader. The emission spectra is 528 nm. The excitation spectra is 480 nm.

Characterization

Aptamer Characterization Protocol

  1. Grow biosensor cells in liquid culture tubes with 5 mL LB and 5 μL antibiotic for 24 hours shaking at 170 RPM at 37°C.
  2. Add 5 μL of culture cells into Erlenmeyer flasks with 50 mL LB and 50 µL antibiotic and grow for an additional 24 hours shaking at 170 RPM at 37°C.
  3. Ensure that the cells are at an OD600 value between 0.4 and 0.8 using a spectrophotometer.
    • If the OD600 value is less than 0.4, grow cells for longer, checking the value every 20-30 minutes.
    • If cells have grown too much, dilute using LB until the concentration matches the desired OD600 value.
  4. Add 3.5 mL of the cell culture into 15 mL liquid culture tubes.
  5. Add 500 µL of diluted IPTG solution (0, 1, 10, 100 µM) .
  6. Grow biosensor cells for an additional 2-4 hours shaking at 170 RPM at 37°C.
  7. Add DFHBI to a final concentration of 200 µM.
  8. Pipette 150 µL of the grown biosensor cells into a well plate to measure cell density (OD600) and fluorescence at excitation wavelength of 492 nm and emission intensity at 501 nm using a plate reader.

Linear DNA Probes Characterization Protocol

  1. Add the following to an amber microcentrifuge tube to make FAM-Probe Mastermix:
    • # of reactions x 1.6 μL= μL Volume of 1 mM FAM Probe
    • # of reactions x 29 μL = μL Volume of TE Buffer (pH 7.5 - 8)
  2. Add the following to a PCR tube:
    • 29.4 μL Mastermix
    • 1.6 μL of 1 mM BHQ-1 Probe
    • 1 μL of diluted complement DNA solution (0, 0.1, 1, 10, 100 μM)
  3. Vortex several seconds and spin down tubes.
  4. Place tubes in thermocycler at 41°C for 1 minute.
  5. Place tubes in thermocycler at 37°C for 1 minute.
  6. Pipette all 32 μL of solution into a 384 well plate to measure fluorescence at excitation wavelength of 480 nm and emission intensity at 518 nm using a plate reader.

Proof of Concept

Overview of Steps

  1. Serum Spiking
  2. miRNA - DNA Hybridization and Ligation
  3. RCA
  4. Readout

1. Serum Spiking

  1. Add the following in a PCR tube:
    • 17.6 μL of pooled human serum
    • 0.4 μL RNase inhibitor
    • 2 μL of 10nM miRNA

2. miRNA - DNA Hybridization and Ligation

  1. Pipette 1 μL of NEB 10 x Ligation Buffer into new PCR tubes.
  2. Add 2.5 μL of 20 nM padlock probe. Add 1 uL of the serum.
  3. Heat the solution at 65°C for 3 minutes, and slowly cool it to room temperature over 10 minutes.
  4. Add 1 μL of Splint R Ligase and 0.5 μL of RNAse inhibitor into the sample.
  5. Incubate the reaction for 2 hours at 37°C.
  6. Bring reaction up to 65°C for 20 minutes to terminate the reaction.

3. RCA Reaction

  1. Pipette 2.5 μL of NEB 10x phi29 buffer into previous ligation reaction mixture.
  2. Add 12.5 units or 1.25 μL of phi29 DNA polymerase. Add 1 μL of aluminum.
  3. Add 6.76 μL of 10 mM dNTPs.
  4. Incubate the PCR tube for 8 hours at 37°C.
  5. Bring the reactions up to 65°C for 10 minutes to inactivate the DNA polymerase.

4. Readout

  1. Add 10 μL of reaction + 2 μL of loading dye into prepared gel.