1.0 Procedure: To perform the INSPECTR ligation through lateral flow (LF) readout protocol, follow each of the steps as described below. See Figures 1 and 2 for workflow and procedure summaries.
1.1. IMPORTANT NOTES
1.1.1. Contamination: CAUTION Precautions must be taken to prevent cross contamination of reaction tubes. Separate work areas should be used for:
· Preparation of master mixes (ligation, rolling circle amplification (RCA), cell-free protein synthesis (CFPS) and lateral flow (LF)) and dilution of DNA oligos
· Target dilution, target addition to ligation, ligation addition to RCA, RCA addition to CFPS, CFPS addition to LF.
1.1.2. Cleaning: CAUTIONTo prevent contamination, ALWAYS clean working surfaces, pipettes, etc. with 20% bleach or other solution that can destroy nucleic acids and RNases. To eliminate accelerated deterioration of any plastics and metals, wipe down with 70% isopropanol after using 20% bleach.
1.2. Thaw RNA target(s): The following steps should be executed in the high-copy lab.
1.2.1. Retrieve target(s) from long term storage:
1.2.1.1. Remove RNA target (from the -80°C freezer) and ExoI (from the -20°C freezer) and place on a cool block.
1.3. Preparation of master mix for ligation: The following steps should be executed in a low target PCR workstation in the main lab.
1.3.1. Retrieve reagents from long term storage:
1.3.1.1. Remove the following materials (from -20°C freezer) and place in a CoolBox: Murine RNase Inhibitor, ET SSB (HC), SplintR Ligase Buffer (or T4 DNA Ligase Buffer), SplintR Ligase (HC), probes for testing, gap filling oligos (GFOs) for testing, dNTP Solution Mix, 10X phi29 DNA Polymerase Buffer, NEB phi29 DNA Polymerase (HC), RCA primers and salmon sperm DNA. Remove the following materials (from room temperature storage) and place on a tube rack at room temperature: PEG 3350.
1.3.1.2. Proceed once all reagents have thawed.
1.3.2. DNA dilution: probes and GFOs may need to be diluted to their working concentrations (10nM for probes, 100nM for GFOs).
1.3.2.1. Pulse vortex the stock tubes at speed 8 out of 10 for 3 seconds and, if necessary, spin down at >100 x g for 3 seconds in a minifuge.
1.3.2.2. For probes (stored at a concentration of 100nM): dilute 1 to 10 (90 uL water + 10 uL of 100nM); pulse vortex at speed 8 out of 10 for 3 seconds.
1.3.2.3. For GFOs (stored at a concentration of 10uM: first dilute 1 to 10 (9 uL water + 1 uL of 10uM) to create a 1uM solution; pulse vortex at speed 8 out of 10 for 3 seconds. Then dilute 1 to 100 (99 uL water + 1 uL of 1uM).
1.3.3. Ligation master mix: Label a 1.5 or 2.0 mL tube “Lig MM”. Prepare the master mix according to Table 1 (scale according to your own Sample ID Table, include an appropriate overage factor). The master mix tube should be kept at room temperature during preparation.
1.3.3.1. Components should be added in the order in which they are listed. For each component (excluding water): pulse vortex the stock at speed 8 out of 10 for 3 seconds and, if necessary, spin down at >100 x g for 3 seconds in a minifuge. When adding components (excluding the first component) to the master mix tube: submerge the pipette tip in the existing liquid and pipette mix twice.
1.3.3.2. After all components are added: pulse vortex the master mix at speed 8 out of 10 for 3 seconds and, if necessary, spin down at >100 x g for 3 seconds in a minifuge.
1.3.3.3. Label strip tubes with the letter L and the ligation reaction number according to your own Sample ID Table and Sample Maps.
1.3.3.4. Aliquot 5 uL of ligation master mix into labeled strip tubes. Do not place on CoolBlock.
1.3.3.5. No target addition: Add 2 uL of water to appropriate ligation reactions according to your own Sample ID Table. Note: This is done in the master mix preparation hood in the main lab so that negative reactions do not need to be opened in a high-copy PCR workstation. Pipette mix 5 times.
1.4. Transfer prepared materials (ligation strip tubes) from the main lab to the high-copy lab using the passthrough.
1.5. Target dilution: Dilute RNA targets according to your own Target Dilution Table. Target dilutions should be done on a CoolBlock. The following steps should be conducted in the target dilution hood (stocked with Low Retention pipette tips).
1.5.1. Add the appropriate volume of water to all tubes according to your own Target Dilution Table.
1.5.2. Pipette mix the stock tube of RNA and add the appropriate volume to the first dilution tube. Pipette mix using this volume twice. Then pipette mix 5 times using a pipette set to the total volume of the dilution.
1.5.2.1. Example: perform a 1 to 10 dilution by adding 1uL of stock RNA to 9uL of water. Pipette mix twice at a volume of 1uL. Then pipette mix 5 times at a volume of 10uL.
1.5.3. Repeat the above for the remaining target dilution tubes.
1.6. Ligation reaction: The following steps should be executed in the high-copy lab.
1.6.1. Target addition: In a PCR workstation, add 2 uL of target to appropriate ligation reactions according to your own Sample ID Table. Pipette mix 5 times.
1.6.2. Spin down at >100 x g for 3 seconds in a minifuge.
1.6.3. Execute ligation protocol on PCR block:
1.6.3.1. 22°C for 35 minutes (no lid heating)
1.6.3.2. 22°C for infinity (no lid heating)
1.7. ExoI digest:The following steps should be executed in the high-copy lab.
1.7.1. Pre-dilute the high concentration ExoI 10-fold, making as much as you need for the number of samples with 10% overage. Each ligation reaction requires 1uL of diluted Exo. Aliquot the diluted ExoI into a strip tube.
1.7.2. Remove the ligation reaction tubes from the PCR block and bring to a PCR workstation. Spin down at >100 x g for 3 seconds in a minifuge. Open ligation reaction tubes with a strip tube opener.
1.7.2.1. Note: Record the time spent in the second 22°C step when you retrieve tubes from PCR block.
1.7.3. Open ExoI enzyme strip tube with a strip tube opener. Add 1 uL of ExoI to each ligation tube. Pipette mix twice at a volume of 1uL. Then pipette mix 5 times at a volume of 8uL. Spin down at >100 x g for 3 seconds in a minifuge.
1.7.3.1. Note:Take care when pipetting ExoI to wipe any excess that accumulates on the pipette tip off inside the enzyme strip tube before adding to the ligation tubes.
1.7.4. Execute ExoI protocol on PCR block:
1.7.4.1. 22°C for 30 minutes (no lid heating)
1.7.4.2. 22°C for infinity (no lid heating)
1.8. Preparation of master mix for RCA: The following steps should be executed in a low target PCR workstation in the main lab.
1.8.1. RCA master mix: Label a 1.5 or 2.0 mL tube “RCA MM”. Prepare the master mix according to Table 2(scale according to your own Sample ID Table, include an appropriate overage factor). The master mix tube should be kept at room temperatureduring preparation.
1.8.1.1. Components should be added in the order in which they are listed. For each component (excluding water): pulse vortex the stock at speed 8 out of 10 for 3 seconds and, if necessary, spin down at >100 x g for 3 seconds in a minifuge. When adding components (excluding the first component) to the master mix tube: submerge the pipette tip in the existing liquid and pipette mix twice.
1.8.1.2. After all components are added: pulse vortex the master mix at speed 8 out of 10 for 3 seconds and, if necessary, spin down at >100 x g for 3 seconds in a minifuge.
1.8.1.3. Label strip tubes with the letter R and the RCA reaction number according to your own Sample ID Table and Sample Maps.
1.8.1.4. Aliquot 6 uL per reaction into labeled strip tubes. Do not place on CoolBlock.
1.9. Transfer prepared materials (RCA strip tubes) from the main lab to the high-copy lab using the passthrough.
1.10. RCA reaction: The following steps should be executed in the high-copy lab.
1.10.1. Add ligation product: Remove the ExoI reaction tubes from the PCR block and bring to a PCR workstation. Spin down at >100 x g for 3 seconds in a minifuge. Open ExoI reaction tubes with a strip tube opener.
1.10.1.1. Note:Record the time spent in the second 22°C step when you retrieve tubes from PCR block.
1.10.2. Pipette mix ExoI product 5 times. Add 6 uL of ExoI product to appropriate RCA reactions according to your own Sample ID Table. Pipette mix 5 times. Spin down at >100 x g for 3 seconds in a minifuge.
1.10.3. Execute RCA protocol on PCR block:
1.10.3.1. 22°C for 2 hours (no lid heating)
1.10.3.2. 22°C for infinity (no lid heating)
1.11. Preparation of reagents for CFPS: The following steps should be executed in a low target PCR workstation in the main lab.
1.11.1. Retrieve reagents from long term storage:
1.11.1.1. When 60 minutes remain in the RCA reaction, remove the following materials (from -80°C freezer) and place in a CoolBox: NEB S30 Extract, 2X Protein Synthesis Buffer, T7 Polymerase and Murine RNase Inhibitor.
1.11.1.2. Proceed once all reagents have thawed.
1.11.2. CFPS master mix: Label a 1.5 or 2.0 mL tube “CFPS MM”. Prepare the master mix according to Table 3 (scale according to your own Sample ID Table -- be sure to account for the peptide controls, include an appropriate overage factor). The master mix tube should be kept on a CoolBlock during preparation.
1.11.2.1. Components should be added in the order in which they are listed. For each component (excluding NEB S30 Extract – pipette mix S30 Extract 10 times to mix): pulse vortex the stock at speed 8 out of 10 for 3 seconds When adding components (excluding the first component) to the master mix tube: submerge the pipette tip in the existing liquid and pipette mix twice. After all components are added: pipette mix 10 times and, if necessary, spin down at >100 x g for 3 seconds in a minifuge.
1.11.2.2. IMPORTANT:NEB S30 Extract should not be vortexed.
1.11.2.3. Label strip tubes with the letter C and the CFPS reaction number according to your own Sample ID Table and Sample Maps.
1.11.2.4. Aliquot 20 uL into labeled strip tubes.
1.11.2.5. Save excess master mix (due to overage). Add the date to the top of the tube. Place in -80°C freezer for later troubleshooting.
1.11.2.6. No template addition: Add 10 uL of water to appropriate CFPS reactions (the conditions that correspond to peptide controls), according to your own Sample ID Table. Note: This is done in the master mix preparation hood in the main lab so that negative reactions do not need to be opened in a high-copy PCR workstation. Pipette mix 5 times.
1.12. Transfer prepared materials (CFPS strip tubes) from the main lab to the high-copy lab using the passthrough.
1.13. CFPS reaction: The following steps should be executed in the high-copy lab.
1.13.1. Add RCA product:Remove the RCA reaction tubes from the PCR block and bring to a PCR workstation. Spin down at >100 x g for 3 seconds in a minifuge. Open RCA reaction tubes with a strip tube opener.
1.13.1.1. Note:Record the time spent in the second 22°C step when you retrieve tubes from PCR block.
1.13.2. Pipette mix RCA product 5 times. Add 10 uL of RCA product to appropriate CFPS reactions according to your own sample ID table. Pipette mix 5 times. Spin down at >100 x g for 3 seconds in a minifuge.
1.13.3. Execute CFPS protocol on PCR block:
1.13.3.1. 22°C for 2 hours (no lid heating)
1.13.3.2. 22°C for infinity (no lid heating)
1.14. Strip-comb preparation: The following steps should be executed in the lateral flow (LF) room of the main lab.
1.14.1. When 60 minutes remain in the CFPS reaction: retrieve the necessary number of strips and cardstock combs according to your own Sample ID Table and Sample Maps.
1.14.2. Place a piece of double-sided tape along a cardstock comb. Pick up a strip (with a gloved hand or tweezers) and carefully place it within the gray shaded region. Gently press on the absorbance pad when the strip is in place. For remaining strips, align such that the bottoms of the strips are at approximately the same height.
1.14.3.1. IMPORTANT:Only handle LF strips by the absorbance pad.
1.14.3.2. Note: If you need to reposition a strip, use tweezers and grab from the bottom of the absorbance pad such that the tweezers hold together the absorbance pad and the strip membrane.
1.14.4. Label the left side of the cardstock comb with the row, according to the your own Sample Mapslayout.
1.15. Preparation of reagents for LF: The following steps should be executed in a low target PCR workstation in the main lab.
1.15.1. Retrieve reagents from long term storage:
1.15.1.1. When 15 minutes remain in the CFPS reaction, remove the following materials (from 4°C fridge) and place on a CoolBox: running buffer and conjugate.
1.15.2. Sonicate conjugate:
1.15.2.1. Turn on Water Bath sonicator (in pre-amp room)
1.15.2.2. Press the "Sonics On/Off" button to turn on sonication.
1.15.2.3. Open tube containing conjugate (if less than 250ul, pool into a new tube)
1.15.2.4. While holding the cap, submerge tube into water bath 3/4 of the way (Ensure conjugate level is below/even with water level). Take care not to spill conjugate into water bath
1.15.2.5. Using a pipette set to around 50% of conjugate volume pipette up and down 5x
1.15.2.6. Turn off machine
1.15.3. LF master mix: Label a 1.5 or 2.0 mL tube “LF MM”. Prepare the master mix according to Table 4(scale according to your own Sample ID Table, include an overage factor of 1.1). The master mix tube should be kept on a CoolBlock during preparation.
1.15.3.1. Components should be added in the order in which they are listed. Pulse vortex the running buffer at speed 8 out of 10 for 3 seconds. Vortex the conjugate at speed 8 out of 10 for 15 seconds. Check the bottom of the conjugate tube. If a pellet of conjugate is present, continue to vortex. Repeat until the pellet has been completely resuspended.
1.16. Transfer prepared materials (LF master mix and prepared cardstock combs) from the main lab to the high-copy lab using the passthrough.
1.17. LF reaction: The following steps should be executed in the high-copy lab.
1.17.1. Vortex the LF master mix at speed 8 out of 10 for 3 seconds. Aliquot 13 uL of LF master mix into wells of a low-bind, flat-bottom reaction plate using an electronic pipette.
1.17.1.1. Note:Do not exceed aspiration and expulsion speeds of 4.
1.17.2. Remove the CFPS reaction tubes from the PCR block and bring to a PCR workstation. Spin down at >100 x g for 3 seconds in a minifuge. Open CFPS reaction tubes with a strip tube opener.
1.17.2.1. Note:Record the time spent in the second 22°C step when you retrieve tubes from PCR block.
1.17.3. Pipette mix CFPS product 5 times. Add 27 uL of CFPS product to appropriate wells according to your own Sample ID Table and Sample Maps. Do not pipette mix.
1.17.4. Creation of controls: Add 1 uL of dual epitope peptide control or water to appropriate wells according to your own Sample ID Table. Do not pipette mix.
1.17.5. Gently swirl plate to mix.
1.17.6. Place a PCR tube rack under the top edge of the plate to create a slight tilt.
1.17.7. Start timer and place strips into appropriate wells. When 20 minutes has passed, proceed to imaging.
1.18. Imaging LF strips
1.18.1. Turn on the computer and the scanner. Open the “EPSON Scan” program.
1.18.1.1. Change Mode to “Professional Mode” if necessary.
1.18.2. Remove cardstock combs from LF plate and place on the flatbed of the scanner.
1.18.4. Click: Preview
1.18.4.1. In Preview dialog box: create a selection to focus on your strips.
1.18.5. Click: Scan