Mike Barnkob:Protocols/CRISPR/Cloning oligos into backbone: Difference between revisions
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Mike Barnkob (talk | contribs) (New page: ← Front page ==Cloning guide sequence into backbone== Protocol for how to clone guide sequences (oligo's) into backbone containing Cas9. Modified from Zhang labs ...) |
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'''Enzymes and reagents:''' | '''Enzymes and reagents:''' | ||
* FastDigest BsmBI ([http://www.thermoscientificbio.com/restriction-enzymes/esp3i-bsmbi/ Thermo Scientific]) | * FastDigest BsmBI (Esp3I) ([http://www.thermoscientificbio.com/restriction-enzymes/esp3i-bsmbi/ Thermo Scientific]) | ||
* 10X FastDigest Buffer (Thermo Scientific) | * 10X FastDigest Buffer (Thermo Scientific) | ||
* FastAP ([http://www.thermoscientificbio.com/dna-and-rna-modifying-enzymes/fastap-thermosensitive-alkaline-phosphatase/ Thermo Scientific]) | * FastAP ([http://www.thermoscientificbio.com/dna-and-rna-modifying-enzymes/fastap-thermosensitive-alkaline-phosphatase/ Thermo Scientific]) | ||
Line 20: | Line 20: | ||
* 2X Quick Ligase Buffer (NEB) | * 2X Quick Ligase Buffer (NEB) | ||
* Quick Ligase (NEB) | * Quick Ligase (NEB) | ||
* ddH20 | * Nuclease free ddH20 | ||
'''Transformation:''' | '''Transformation:''' | ||
* Stbl3 bacteria | * Stbl3 bacteria [https://www.lifetechnologies.com/order/catalog/product/C737303 Life Technologies] | ||
===Protocol=== | ===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: | |||
# Add | #* 2 μL 10x FastAP buffer | ||
# | #* 1 μL FastAP (1 units) | ||
# Spin | # 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 | ||
# Add | # Centrifuge briefly for a few seconds | ||
# | # Ligate: let reaction sit for 10 min at room temperature. | ||
# | # Chill on ice | ||
# | |||
# | |||
'''Quality control:' | '''Quality control:''' | ||
# Check product by running 1 μL on gel. If many bands are showing up consider gel purification. | |||
# | |||
'''Storage:''' | '''Storage / next steps:''' | ||
# Then either store at -20°C or move on to transformation into bacteria. <br />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. <br />Note: Only use RecA- bacteria, such as Stbl3, since construct contain LTRs. | |||
===References=== | ===References=== | ||
* | pLentiCRISPRv2 protocol: | ||
* | * Addgene: https://www.addgene.org/static/data/plasmids/52/52961/52961-attachment_B3xTwla0bkYD.pdf | ||
* | |||
* | Ligation: | ||
* https://www.neb.com/products/m2200-quick-ligation-kit | |||
Digest: | |||
* http://www.thermoscientificbio.com/restriction-enzymes/esp3i-bsmbi/ | |||
* http://www.thermoscientificbio.com/dna-and-rna-modifying-enzymes/fastap-thermosensitive-alkaline-phosphatase/ | |||
Annealing oligos: | |||
* http://www.sigmaaldrich.com/technical-documents/protocols/biology/annealing-oligos.html | |||
* https://www.addgene.org/plasmid-protocols/annealed-oligo-cloning/ |
Revision as of 07:28, 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
Transformation:
- Stbl3 bacteria Life Technologies
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: