Cas3 Roadblocks-RNAP

Contents

1. Imaging buffer
2. Protocol
3. Preparation of the first injection
4. Injections
5. Injection protocol

1. Imaging buffer

Use default roadblocks conditions + NTPs.

• 40 mM Tris
• 0.2 mg/ml BSA
• 2 mM MgCl2
• 50 mM NaCl
• 1 mM ATP
• 1:1000 saturated biotin solution
• 25 uM of GTP and UTP (250 mM stocks in green and orange tubes in max‘s 4’ box)

Caution

Only add NTPs to buffer you are using. I add 2 uL stock to 20 ml imaing buffer for each experiment, collecting the leftovers and adding it next rounds imaging buffer

Caution

Omit CTP to generate stalled condition

2. Protocol

1. Add 10 nM Cascade final to 50 uL of WT NEB lambda DNA.
2. Incubate at 55’ for ten minutes.
3. Cool off to room-temp for ten minutes.
4. Dilute to 1 ml injection and inject as normal.
5. Prepare the first injection.

3. Preparation of the first injection

1. 1 uL RNAP (1:20 dilution) + 1 uL strep QD 605.
2. 1 uL anti-FLAG antibody (1:10 dilution) + 1 uL strep QD 705.
3. Incubate reactions separately for 20 minutes.
4. After incubation, add 3 uL of saturated biotin solution to each reaction.
5. Incubate separately for 10 minutes.
6. Mix the reactions, and dilute with 150 uL Imaging buffer.

4. Injections

1. Injection 1 as prepared above.
2. 960 uL imaging buffer + 40 ul heparin (5 mg/ml). (In max‘s 4’ box)
3. 1.5 uL Cas3 into 150 uL Imaging buffer.

5. Injection protocol

1. Injection 1 is at 100 ul/min to promote binding. You can go faster until it reaches the flowcell. Wash it out after you can semi-see through the cloud.
2. Inject injection 2 in the 800 uL loop at 500 ul/min for 1 minute. Then close the loop by turning to load ‘position 2’. WAIT 2 MINUTES bofore injecting Cas3. Otherwise, heparin may inhibit Cas3.
3. Injection 3 is at 500 ul/min.