Mike Barnkob:Protocols/CRISPR/Cloning oligos into backbone

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Cloning guide sequence into backbone

Protocol for how to clone guide sequences (oligo's) into backbone containing Cas9. Modified from Zhang labs (MIT) protocol.

Reagents and setup

DNA:

  • Backbone plasmid
  • Guide oligo's

Enzymes and reagents:

  • FastDigest BsmBI (Esp3I) (Thermo Scientific)
  • 10X FastDigest Buffer (Thermo Scientific)
  • FastAP (Thermo Scientific)
  • 100 mM DTT (Sigma)
  • 10X T4 Ligation Buffer (NEB)
  • T4 PNK (NEB)
  • 2X Quick Ligase Buffer (NEB)
  • Quick Ligase (NEB)
  • Nuclease free ddH20

Setup:

  • Besides cloning gRNA's into the backbone, additionally, prepare one uncut backbone (positive control) and one backbone that is cut, but has nothing ligated into it (negative control).

Protocol 1 (Yale style)

Digest backbone:

  1. Prepare the following:
    • 100mM DTT: 1 μL 1M DTT into 9 μL ddH20.
    • Turn on water-bath to 37°C
  2. Add the following in order to Eppendorf tube:
    • x μL dH20 (up to 20 μL)
    • 2 μL 10X FastDigest Buffer
    • 2 ug pLentiCRISPRv2 backbone plasmid
    • 0.6 μL 100mM DTT
    • 1 μL (10 units) FastDigest BsmBI
  3. Gently mix by pipetting
  4. Spin tube down for few seconds
  5. Digest by incubating in waterbath at 37°C for 90 minutes

Dephosphorylate:

  1. Add the following to the digested backbone:
    • 2 μL 10x FastAP buffer
    • 1 μL FastAP (1 units)
  2. Gently mix by pipetting
  3. Spin tube down briefly
  4. Incubate in waterbath at 37°C for 1 hour
  5. Stop reaction by incubating at 75°C for 5 minutes.
  6. Measure concentration on Nanodrop

Anneal oligos:

  1. Prepare the following:
    • Dilute primers to 100 μM (100pmol/uL) in new tubes
    • The negative control, using H20 instead of anneal oligo's.
  2. Add the following in a PCR tube:
    • 6.5 μL ddH20
    • 1 μL oligo 1 (100 μM)
    • 1 μL oligo 2 (100 μM)
    • 1 μL 10X T4 DNA Ligation Buffer
      Note: Use the T4 DNA Ligation buffer instead of the buffer that comes with the enzyme, as this buffer contains ATP needed by the enzyme.
    • 0.5 μL T4 PNK
  3. Place in thermal cycler and run using the following program:
    • Step 1: 37°C for 30 min
    • Step 2: 95°C for 5 min
  4. Remove tubes and let tubes sit at room temperature for 50 minutes.
  5. Briefly spin tubes to draw all moisture from the lid
  6. Dilute annealed oligos at a 1:200 dilution into sterile water.
  7. Keep on ice or 4°C

Ligate annealed oligo into backbone:

  1. Mix the following together in 1.5 Eppendorf tube:
    • 50ng digested plasmid backbone
    • 1 μL of diluted, annealed oligos
    • 5 μL 2X Quick Ligase Buffer
    • Add ddH20 up to 10 μL
    • 1 μL Quick Ligase
  2. Mix gently by pipetting
  3. Centrifuge briefly for a few seconds
  4. Ligate: let reaction sit for 10 min at room temperature.
  5. Chill on ice

Quality control:

  1. Check product by running 1 μL on gel. If many bands are showing up consider gel purification.

Storage / next steps:

References

pLentiCRISPRv2 protocol:

Ligation:

Digest:

Annealing oligos: