Mike Barnkob:Protocols/CRISPR/Cloning oligos into backbone: Difference between revisions
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* Quick Ligase (NEB) | * Quick Ligase (NEB) | ||
* Nuclease free ddH20 | * Nuclease free ddH20 | ||
===Protocol 1 (Yale style)=== | ===Protocol 1 (Yale style)=== |
Revision as of 07:29, 29 January 2015
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
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
Protocol 1 (Yale style)
Digest backbone:
- Prepare the following:
- 100mM DTT: 1 μL 1M DTT into 9 μL ddH20.
- Add the following in order to Eppendorf tube:
- 15.5 μL dH20
- 2 μL 10X FastDigest Buffer
- 2 ug pLentiCRISPRv2 backbone plasmid
- 0.6 μL 100mM DTT
- 1 μL (10 units) FastDigest BsmBI
- Gently mix by pipetting
- Spin tube down for few seconds
- Digest by incubating in waterbath at 37°C for 90 minutes
- Measure concentration on Nanodrop
Dephosphorylate:
- Add the following to the digested backbone:
- 2 μL 10x FastAP buffer
- 1 μL FastAP (1 units)
- Gently mix by pipetting
- Spin tube down briefly
- Incubate in waterbath at 37°C for 1 hour
- Stop reaction by incubating at 75°C for 5 minutes.
Anneal oligos:
- Prepare the following:
- Dilute primers to 100 μM in new tubes
- Add the following in a PCR tube:
- 6.5 μL ddH20
- 1 μL oligo 1 (100 μM)
- 1 μL oligo 2 (100 μM)
- 10X T4 Ligation Buffer
- 0.5 T4 PNK
- Place in thermal cycler and run using the following program:
- Step 1: 37°C for 30 min
- Step 2: 95°C for 5 min
- Step 3: Decrease 5°C every minute until at 25°C
- Briefly spin tubes to draw all moisture from the lid
- Put on ice or 4°C
- Dilute annealed oligos at a 1:200 dilution into sterile water.
Ligate annealed oligo into backbone:
- 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
- Mix gently by pipetting
- Centrifuge briefly for a few seconds
- Ligate: let reaction sit for 10 min at room temperature.
- Chill on ice
Quality control:
- Check product by running 1 μL on gel. If many bands are showing up consider gel purification.
Storage / next steps:
- Then either store at -20°C or move on to transformation into bacteria.
Note: Only use RecA- bacteria, such as Stbl3, since construct contain LTRs.
Protocol 2 (a la Thermo Scientific style)
Anneal oligos:
- Add the following in a PCR tube:
- 6.5 μL ddH20
- 1 μL oligo 1 (100 μM)
- 1 μL oligo 2 (100 μM)
- 10X T4 Ligation Buffer
- 0.5 T4 PNK
- Place in thermal cycler and run using the following program:
- Step 1: 37°C for 30 min
- Step 2: 95°C for 5 min
- Step 3: Decrease 5°C every minute until at 25°C
- Dilute annealed oligos at a 1:200 dilution into sterile water.
Digest backbone and dephosphorylate
- Add the following in order to Eppendorf tube:
- 8 μL dH20 (up to 20 μL total reaction)
- 2 μL 10X FastDigest Buffer
- 2 ug pLentiCRISPRv2 backbone plasmid
- 1 μL (10 units) FastDigest BsmBI
- 1 μL (1 unit) FastAP
- Gently mix by pipetting
- Spin tube down briefly
- Digest by incubating in waterbath at 37°C for 10 minutes
- Inactivate enzymes by incubating at 80°C for 20 minutes
Ligate annealed oligo into backbone:
- 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
- Mix gently by pipetting
- Centrifuge briefly for a few seconds
- Ligate: let reaction sit for 10 min at room temperature.
- Chill on ice
Quality control:
- Check product by running 1 μL on gel. If many bands are showing up consider gel purification.
Storage / next steps:
- Then either store at -20°C or move on to transformation into bacteria.
Note: Only use RecA- bacteria, such as Stbl3, since construct contain LTRs.
References
pLentiCRISPRv2 protocol:
Ligation:
Digest:
- http://www.thermoscientificbio.com/restriction-enzymes/esp3i-bsmbi/
- http://www.thermoscientificbio.com/dna-and-rna-modifying-enzymes/fastap-thermosensitive-alkaline-phosphatase/
Annealing oligos: