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Experiments

LB [Ampicillin] Agar Preparation

1. 35g of LB agar was added into 1L of distilled water in a 2L flask.
2. Solution was autoclaved for 15 minutes at 121 °C.
3. 1000µL of ampicillin (100mg/mL) was added to reach a final concentration of 100µg/mL

LB Broth Preparation

1. 25g of LB broth was added into 1L of distilled water in a 2L flask.
2. Solution was autoclaved for 15 minutes at 121 °C.
3. 1000µL of ampicillin (100mg/mL) was added to reach a final concentration of 100µg/mL.

Inoculation

1. Pipette 6 mL of LB Broth into each tube.
2. Use an inoculation loop to take a colony from the agar plate and transfer the colony to the tubes containing LB Broth.
3. Incubate the tubes at a temperature of 37°C in a shaker incubator at 225 RPM for 18-24hrs.

Preparation of gBlock (IDT DNA protocol for gBlock)

1. Prior to opening, centrifuge the tube at a minimum of 13.3 rpm for 1 minute to ensure that the material is at the bottom of the tube.
2. Add appropriate volume of TE buffer to the lyophilized gBlock to reach a final concentration of 100ng/µl or 50ng/µl.
3. Vortex the sample for 5-10 seconds.
4. Incubate in the Eppendorf heat block at 50°C for 20 minutes.
5. Vortex the sample for 5-10 seconds.
6. Centrifuge the sample for 1 minute.
7. Store at -20˚C.

Preparation of PCR Primers

1. Prior to opening, centrifuge the tube at 13.3 rpm for 1 minute to ensure that the material is at the bottom of the tube.
2. Add x volume of TE buffer to reach a final concentration of 100µM for each primer according to manufacturer specification.
3. Mix the tubes by pipetting and place on ice.
4. Dilute the 100µM stock solution to a working solution of 10µM at a final volume of 50µL.

PCR Protocol

1. Preheat the thermocycler to 95 °C.
2. Allow the reagents to thaw in ice.
3. Prepare the PCR tubes with the following mixture composition:
• - 12.5µL of NEB Q5 High-Fidelity 2X Master Mix
• - 1.25µL of forward primer (10μM)
• - 1.25µL of reverse primer (10μM)
• - 2µL of DNA template
• - 8µL of nuclease-free water
4. Spin the tubes for a few seconds.
5. Transfer the tubes to the thermocycler and set the following temperatures and settings:
• - Initial denaturation: 98°C for 30 seconds
• - 30 cycles:
1. Denaturation: 98°C for 10 seconds
2. Annealing: 50-72˚C for 30 seconds (Annealing temperature selected based on Tm of primer set; typically 3-5˚C below the Tm)
3. Extension: 72°C for 15 seconds
• - Final Extension: 72°C for 2 minutes
• - Hold: 4°C
6. Tubes stored at -20˚C or ran on gel electrophoresis immediately post PCR-amplfiication.

1. Prepare the ligation reaction in a PCR tube on ice.
2. Add 10µL of 2X reaction buffer to the PCR tube.
3. Add 1µL of non-purified PCR product.
4. Add 1µL of pJET1.2/blunt cloning vector.
5. Add 7µL of nuclease free water.
6. Add 1µL of T4 DNA ligase.
7. Vortex for 5 seconds.
8. Centrifuge for 5 seconds.
9. Incubate the ligation mixture in the PCR machine for 5 minutes at 22˚C.
10. . Use mixture directly after step 9 for transformation.

1. Thaw a tube of NEB 5-alpha Competent *E. coli* cells on ice for 10 minutes.
2. Add 1-5µL containing 1pg - 100ng of plasmid DNA to the cell mixture.
3. Carefully flick the tube 4-5 times to mix cells and DNA (do not vortex).
4. Place the mixture on ice for 30 minutes.
5. Heat shock at exactly 42˚C for exactly 30 seconds.
6. Place on ice for 5 minutes.
7. Pipette 950µL of room temperature SOC into the mixture.
8. Place at 37˚C for 60 minutes.
9. Shake vigorously (225 rpm) or rotate.
10. . Warm selection plates to 37˚C during the 60 minute incubation from steps 8 & 9.
11. . Centrifuge the tubes at 13.3 rpm for 1 minute.
12. . Remove 850µL of the supernatant and re-suspend the cells with the remaining supernatant in order to concentrate them.
13. . Spread the remaining 55µL concentrated cells on appropriately labeled agar plates along with the respective antibiotic.
14. . Incubate at 37˚C for 18-24hrs.

Miniprep

1. Pellet 1-5mL bacterial overnight culture by centrifugation at >8000 rpm for 3 min at room temperature.
2. Discard the supernatant.
3. Resuspend pelleted bacterial cells in 250µL resuspension buffer and transfer to a microcentrifuge tube.
4. Add 250µL Buffer lysis buffer and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear (do not allow the lysis reaction to proceed for more than 5 min).
5. Add 350µL Buffer neutralization buffer and mix immediately and thoroughly by inverting the tube 4-6 times.
6. Centrifuge for 10 min at 13,000 rmp.
7. Apply 800µL supernatant to the QIAprep 2.0 spin column by pipetting.
8. Centrifuge for 30-60 seconds and discard the flow-through.
9. Wash the QIAprep 2.0 spin column by adding 0.5mL binding buffer.
10. . Centrifuge for 30-60 seconds and discard the flow-through.
11. . Wash the QIAprep 2.0 spin column by adding 0.75mL wash buffer.
12. . Centrifuge for 30-60 seconds and discard the flow-through.
13. . Centrifuge for 1 minute to remove residual wash buffer.
14. . Place the QIAprep 2.0 column in a clean and appropriately labelled 1.5mL microcentrifuge tube in order to elute the plasmid.
15. . Add 50µL elution buffer to the center of the QIAprep 2.0 spin column.
16. . Let stand for 1 minute and centrifuge for 1 minute.

Nanodrop

1. Blank the nanodrop spectrophotometer using water or elution buffer according to the solution used to elute plasmid DNA from the miniprep protocol.
2. Measure DNA concentration using the nanodrop by taking a 2µL sample of the plasmid.
3. Wipe the sample from both the upper and lower pedestal after each measurement using Kim Wipes and repeat for additional samples.

Making gels

1. Mix 40mL of 50X TAE buffer with 1960mL of MilliQ water to make 2L of 1X TAE buffer.
2. Prepare 1% or 3% agarose gels by weighing out appropriate amount of agarose according to concentration of gel required in a final volume of 50mL of 1X TAE buffer (e.g. 0.5grams used for 1% agarose gel).
3. Microwave mixtures in increments until boiling to dissolve the Agarose.
4. Add 3µl of Gel green (final concentration of 5x) into the 50mL agarose mixture.
5. Pour the solution into the casting tray and place the well combs.
6. Let the gel solidify and remove comb prior to loading.

Loading Gels

1. Add the appropriate amount of 5x loading dye to the set volume of DNA to obtain a 1x concentration of the loading dye (e.g. 1 µL of loading dye to 5 µL of DNA).
2. Place set gel into electrophoresis chamber.
3. Load 5µl of the ladder and each of the samples into their appropriate wells.
4. Pour 1X TAE buffer carefully into the chamber to submerge the gel.
5. Connect the electrophoresis chamber to MyRun electrophoresis.
6. Run gel on 135 V for 20 minutes.
7. Visualize the gel using UV light or E-gel imager using Blue Light base.

RPA Protocol

1. Resuspend RPA primers to final concentration of 100µM.
2. Make a 5µM dilution for each primer for RPA reactions.
3. Prepare the reaction mix in 1.5mL tube:
• - Primer Forward (5µM) - 2.4µL
• - Primer Reverse (5µM) - 2.4µL
• - Rehydration Buffer - 29.5µL
• - dH2O - 8.2µL
4. Add the reaction mix (42.5µL) to 1 freeze dried RPA provided tube, and mix by pipetting up and down.
5. Split the reaction into 2 volumes of 15µL into 2 separate PCR tubes.
6. Add 1µL of 280mM magnesium acetate to each tube and mix well to start the reaction.
7. Add 5µL of DNA to one tube and 5µL of nuclease-free water to the negative control tube.
8. Incubate 20 minutes at 37°C.

DETECTR RPA CRISPR Cas12a Protocol

1. Add the following reagents in this order at the room temperature:
• - 2µL NEBuffer 2.1 Reaction Buffer (10x)
• - 0.5µL 5µM gRNA
• - 1.5µL 1 µM EnGen Lba Cas12a (Cpf1)
2. Make the RPA reactions on target gene to obtain a total volume of 21µL.
3. Incubate both CRISPR complex for 10 minutes at 37°C.
4. Add 1µL of 50µM FQ quencher to the CRISPR complex.
5. Add CRISPR complex mix (5µL) with quencher to RPA reaction mix.
6. Incubate 30 minutes at 37°C.
7. Check under the blue light based E-gel imager.

• LB [Ampicillin] Agar Preparation
• LB Broth Preparation
• Inoculation
• Preparation of gBlock
• Preparation of PCR Primers
• PCR Protocol
• Ligation
• Transformation
• Miniprep
• Nanodrop
• Making gels
• Loading gels
• RPA Protocol
• DETECTR RPA CRISPR Cas12a Protocol

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