Rolling Circle Amplification (RCA)

RCA | 2022 Protocol

DNA-RNA Hybridization

1. Heat the solution at 65°C for 3 minutes, and slowly cool it to room temperature over 10 minutes.

Ligation

1. Incubate the reaction for 2 hours at 37°C.
2. Bring the reaction up to 65°C for 20 minutes to terminate.

Amplification

1. Incubate the reactions for 8 hours at 37°C.
2. Bring the reactions up to 65°C for 10 minutes to inactivate the DNA polymerase.

miRNA Detection Limit Experimentation | 2022 Protocol

Optimized RCA

DNA-RNA Hybridization

1. Heat the solution at 65°C for 3 minutes, and slowly cool it to room temperature over 10 minutes.

Ligation

1. Incubate the reaction for 30 minutes at 37°C.
2. Bring the reaction up to 65°C for 20 minutes to terminate.

Amplification

1. Incubate the reactions for 5 hours at 37°C.
2. Bring the reactions up to 65°C for 10 minutes to inactivate the DNA polymerase.

Amplification with Phi29-XT

Amplification

1. Incubate the reaction for 3 minutes at 95°C with the lid set at 100°C.
2. Incubate the reaction for 2 hours at 42°C.
3. Bring the reactions up to 65°C for 10 minutes to inactivate the DNA polymerase

BlueGel™ with RCP | 2022 Protocol

1. For a 2% agarose concentration, add 0.4 g of agarose to 20 mL of 1X BE buffer.
2. Microwave gel mix for 30 seconds, or until clear.
3. Allow the solution to cool until 65°C.
4. Add 2 uL of SYBR™ Safe.
5. Pour gel mixture into gel mold with combs.
6. Let cool at room temperature and set.

Readout Reaction

1. Add 10 μL of RCP + 2 μL of loading dye into the gel
2. Add 12uL of the 2-log ladder into the gel
3. Start the blueGel™ gel electrophoresis system.
4. After 1 hour, use the UV illumination on the blueGel™ gel electrophoresis system to visualize the DNA.

Linear DNA Probes with RCP | 2022 Protocol

Add the following to an amber microcentrifuge tube to make FAM-Probe Mastermix:

In nuclease-free PCR tubes add the following triplicates:

1. Flick and spin down tubes.
2. Place tubes in the thermocycler at 41°C for 1 minute.
3. Place tubes in thermocycler 37°C for 1 minute.
4. Pipette all 32 μL of solution into a 384-well plate to measure fluorescence at an excitation wavelength of 480 nm and emission intensity at 518 nm using a plate reader.

Lettuce with RCP | 2022 Protocol

In nuclease-free PCR tubes add the following triplicates:

1. Centrifuge the reagents.
2. On the sides of the centrifuged PCR tubes, carefully add 1.5 μL of DFHBI-1T (100 uM stock)
3. For controls without the dye, 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, and the excitation spectra is 480 nm.

SYBR™ Safe with RCP

1. Vortex and centrifuge reagents.
2. Incubate the reaction at 37°C for 1 hour.
3. Read fluorescence on the plate reader. The emission spectra is 528 nm, and the excitation spectra is 480 nm.

Capillary RCA

DNA-RNA Hybridization

1. Heat the solution at 65°C for 3 minutes, and slowly cool it to room temperature over 10 minutes.

Ligation

|1 uL SplintR Ligase 0.5 uL RNase Inhibitor|

1. Incubate the reaction for 2 hours at 37°C.

2. Bring the reaction up to 65°C for 20 minutes to terminate.

3. Orient PCR tubes horizontally and slide capillary tubes into each tube.

4. Allow capillary action to draw up reaction mixture until there is about 1 centimeter of air left in the tube.

5. Carefully slide the capillary tubes into the sigillum wax tray on both ends of the tube.

6. Apply a thin coat of transparent nail polish to both sides of the capillary tube, and let dry for approximately 30 seconds.

7. Place the capillary tubes in a tray and incubate at 37°C for 5 hours.

8. Incubate the remainder of the reaction mixture in PCR tubes at 37°C for 5 hours.

9. Carefully remove the tray from the incubator, and place the tray into a UV illuminator.

10. Image the tubes with a phone camera and count the number of dots visualized in each capillary tube.

GEL:

1. Use an air pump to blow the liquid from the capillary tubes into PCR tubes.
2. Add 2 uL loading dye to the PCR tubes and pipette into gel.
3. For the RCA reactions run within PCR tubes:
4. Pipette 10 uL of RCP + 2 uL loading dye into the gel.
5. Add 12uL of the 2-log ladder into the gel.
6. Start the blueGel™ gel electrophoresis system.
7. After 1 hour, use the UV illumination on the blueGel™ gel electrophoresis system to visualize the DNA

eRCA Protocol

DNA-RNA Hybridization

1. Heat the solution at 65°C for 3 minutes, and slowly cool it to room temperature over 10 minutes.

Ligation

1. Incubate the reaction for 2 hours at 37°C.
2. Bring the reaction up to 65°C for 20 minutes to terminate.

Amplification

1. Incubate the reactions for 5 hours at 37°C
2. Bring the reactions up to 65°C for 10 minutes to inactivate the DNA polymerase

eRCA Readout

In nuclease-free PCR tubes add the following triplicates:

1. Centrifuge the reagents.
2. On the sides of the centrifuged PCR tubes, carefully add 1.5 μL of DFHBI-1T (100 uM stock)
   • For controls without the dye, steps 1-3 were repeated with 1.5 μL RNase-free water being added in place of the dye.
3. Vortex and centrifuge reagents.
4. Incubate the reaction at 70°C for 5 minutes, then cool to and incubate at 25°C for 1 hour.
5. Read fluorescence on the plate reader. The emission spectra is 528 nm, and the excitation spectra is 480 nm.

Protein Purification

NEBExpress Ni Spin Column Protocol

To prepare buffers for Ni Spin Columns under Native Conditions

Column Preparation

1. Remove the bottom tab of the column by twisting, loosen the top cap and place the column in the collection tube provided.
2. Centrifuge column at 800 x g for 1 minute to remove the storage buffer, discard the buffer.
3. Add 250 μl of Lysis/Binding buffer to the column.
4. Centrifuge column at 800 x g for 1 minute, discard the Lysis/Binding buffer.
5. Place the column in a new 2 ml microcentrifuge tube

Sample Cell Lysis

1. Add 5ml of Luria Broth (LB) to a culture tube
2. Add 5ul of antibiotic to the culture tube
3. Collect a colony from the transformation plate using a pipette tip and insert it into the culture tube
4. Let the tube shake in the incubator and allow growth for 24 hours
5. Moved 2 ml of inoculation culture to a microcentrifuge tube
6. Centrifuged the microcentrifuge tube for 5 min at 15600 rpm
7. Removed supernatant (liquid LB) from the centrifuge tube, leaving only the cell pellet
8. Add 250μL of TE buffer to the microcentrifuge tube containing the cell pellet
9. Hydrated lyophilized lysozyme with 1mL TE buffer
10. Added 50μL of lysozyme into the microcentrifuge tube for the final protein sample lysate

Lysate Binding

1. Add up to 500 μl of the protein sample lysate to the column. Note: If sample volume is greater than 500 μl multiple applications can be performed; collect the flow through in separate microcentrifuge tubes.
2. Tap the column to mix the lysate with the resin and allow binding for 2 minutes. Note: Binding of some His-tagged proteins can be increased with longer mixing times. Cap the column and seal the bottom using the plug provided. Mix end-over-end at 4°C for desired time (typically 5-15 minutes). Prolonged mixing may result in more non-specific binding.
3. Centrifuge column at 800 x g for 1 minute, reserve flow through.
4. Place the column in a new 2 ml microcentrifuge tube.

Column Wash

1. Add 250 μl of Wash Buffer to the column and centrifuge at 800 x g for 1 minute.
2. Repeat wash step twice, collect each wash in a separate 2 ml microcentrifuge tube.

Protein Elution

1. Place column in a new 2 ml microcentrifuge tube.
2. Add 200 μl of Elution Buffer to the column. Mix the resin with the elution buffer thoroughly. Note: Elution volume can be reduced to 100 μl if a more concentrated protein sample is desired, total target protein yield may be lower.
3. Centrifuge at 800 x g for 1 minute, save eluted sample.
4. Place column in a new 2 ml microcentrifuge tube and repeat elution step. Typically, >90% of the bound protein is eluted following the second elution.
5. Analyze the clarified cell lysate (load), flow through, washes and eluates by SDS-PAGE.