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Finkelstein Lab Wiki

Cas3 Roadblocks-LacI

Contents

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

1. Preparation

Section titled “1. Preparation”
  • Need to make and purify PBamp lysogen #386
  • Use the lambda DNA purification protocol
  • Use LacI in BB10.4c (11 uM). Colored tubes are stocks. Clear tubes are 10 uL aliqots.
  1. Make a working dilution of LacI (63 nM) by diluting 2 uL of stock into 350 uL BSA buffer - DTT.

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’ 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.