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Finkelstein Lab Wiki

Cas3 Roadblocks-LacI

For the RNA polymerase roadblock assay, see Cas3 Roadblocks-RNAP. For protein purifications used in this assay, see 3xFLAG Lac Repressor (LacI) Purification, T. fusca Cascade Purification, and T. fusca Cas3 Purification.

Contents

Section titled “Contents”
  1. Preparation
  2. Imaging buffer
  3. Protocol
  4. Injections

1. Preparation

Section titled “1. Preparation”

2. Imaging buffer

Section titled “2. Imaging buffer”

Use default roadblocks conditions.

  • 40 mM Tris
  • 0.2 mg/ml BSA
  • 2 mM MgCl2
  • 50 mM NaCl
  • 1 mM ATP
  • 1:1000 saturated biotin solution

3. Protocol

Section titled “3. Protocol”
  1. Add 10 nM Cascade final to 50 uL of site B cassette lambda DNA.
  2. Incubate at 55°C for ten minutes.
  3. Cool off to room-temp for five minutes.
  4. Add 2 uL of LacI (63 nM) to the DNA and incubate on ice for 5 minutes.
  5. Dilute to 1 ml injection and inject as normal.

4. Injections

Section titled “4. Injections”
  • Injection 1: 1 uL anti-HA antibody (1:10 dilution) + 1 uL anti-rabbit QD 705 + 1 uL anti-FLAG antibody (1:10 dilution) + 1 uL strep QD 605.
    1. Incubate 10 minute and dilute into 150 uL Imaging buffer.
  • Injection 2: 1.5 uL Cas3 into 150 uL Imaging buffer.