On-device miSHERLOCK reactions

miSHERLOCK protocol

Setting up the device:

1 -3D print the desired miSHERLOCK device and components (heater platform, device housing, saliva collector, plunger, and sample preparation column) using black resin. Files for the duplex, triplex, and quadruplex devices can be found here. 

2 - Assemble the temperature control, LED, and temperature circuits following the circuit diagram provided, which  can be found in Fig. S15 here. 

3 - Attach heaters to the heater platform and insert the LEDs and temperature control circuit into the miSHERLOCK housing. Images of how to correctly insert them can be found in Fig. S16 here.

4 - Cut the 2mm orange acrylic for the transilluminator ( 2.75 cm × 2.25 cm, 2.75 cm × 3.2 cm, or 2.75 cm × 4.0 cm for the duplex, triplex, or quadruplex transilluminator filter, respectively)  

4 - Attach the 4mm PES filter to the bottom of the sample preparation column.

5 - Insert Whatman gel blotting paper to the correct region on the heater platform.

6 - Line the inside of the reservoir on the miSHERLOCK device housing  with double-sided tape and attach foil to the underside of the reservoir, making sure that it is fully sealed.

7 - Fill each reservoir with 50uL nuclease-free water.

8 - insert new SHERLOCK assays to the bottom of the chamber.

9 - Connect heaters to the battery power when ready to use.

Lyophilized one-pot SHERLOCK reaction 

Prepare gRNA: 

1 - Combine the following components: 

1 - 1uL of ssDNA sequence to be transcribed into RNA (with T7 promoter sequence) from a 50uM stock concentration 

2 - 0.55uL of complimentary T7 promoter sequence from a 100uM stock concentration 

3 - 0.5uL of NaCl (5M) 

4 - 47uL TE buffer

2 - Heat the following solution to 95C for 5 minutes then allow to cool to room temperature  

3 - Prepare a HiScribe Quick Reaction with the following components 

    1 - 8 uL of annealed DNA solution 

    2 - 2uL of T7 RNA polymerase mix 

    3 - 10uL NTP buffer mix 

    4 - 10uL nuclease free water 

4 - Incubate for 18 hours at 37C

5 - Perform RNA cleanup and concentration using Zymo commercial kit 

Prepare lyophilized reaction: 

1 - Prepare one-pot SHERLOCK solution using the following components into a 0.2mL PCR tube and mix well: 

    1 - TwistAmp Basic pellet 

    2 - 0.24uL of forward and reverse RPA primers each from a 100uM stock concentration 

    3 - 30uL of rehydration buffer (33mM HEPES pH 6.8, 100mM NaCl, 8.3% PEG) 

    4 - 2.5uL of Protoscript reverse transcriptase from a 100uM stock concentration

    5 - 0.25uL of Ambion RNase H from a 10U/uL stock concentration 

    6 - 0.5uL of ssDNA probe 

    7 - 0.2uL of EnGen Lba Cas12a from a 50uM stock concentration 

    8 - 0.2uL of gRNA solution from a 100uM stock concentration 

    9 - 5uL of 10x NEB 2.1 buffer

2 - Punch a hole into the 0.2mL PCR tube cap using an 18 Gauge needle 

3 - Snap freeze the above PCR tube in liquid nitrogen 

4 -  Quickly re-open snap-frozen PCR tube and add 2.5uL of Magnesium acetate from a 280mM stock concentration 

5 - Snap freeze the PCR tube again 

6 - Lyophilize the snap-frozen PCR tube for 6 hrs 

Run miSHERLOCK assays

1 - Load lyophilized 0.2mL PCR one-pot SHERLOCK assay into miSHERLOCK device constructed above 

2 - Add 2mL unprocessed saliva added to saliva concentrator and turn on miSHERLOCK device for sample preparation chamber to be heated to 95C. Saliva should filter by gravity through sample preparation chamber into waste filter 

3 - Transfer sample preparation chamber , resuspend the pellet and run for 1h .

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