Mike Barnkob:Protocols/CRISPR/Cloning oligos into backbone: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
No edit summary
No edit summary
 
(8 intermediate revisions by the same user not shown)
Line 5: Line 5:
Protocol for how to clone guide sequences (oligo's) into backbone containing Cas9. Modified from Zhang labs (MIT) protocol.
Protocol for how to clone guide sequences (oligo's) into backbone containing Cas9. Modified from Zhang labs (MIT) protocol.


===Reagents===
===Reagents and setup===


'''DNA:'''
'''DNA:'''
Line 22: Line 22:
* Nuclease free ddH20
* Nuclease free ddH20


'''Transformation:'''
'''Setup:'''
* Stbl3 bacteria [https://www.lifetechnologies.com/order/catalog/product/C737303 Life Technologies]
* 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)===
===Protocol 1 (Yale style)===
Line 30: Line 30:
# Prepare the following:
# Prepare the following:
#* 100mM DTT: 1 μL 1M DTT into 9 μL ddH20.
#* 100mM DTT: 1 μL 1M DTT into 9 μL ddH20.
#* Turn on water-bath to 37°C
# Add the following in order to Eppendorf tube:
# Add the following in order to Eppendorf tube:
#* 15.5 μL dH20
#* x μL dH20 (up to 20 μL)
#* 2 μL 10X FastDigest Buffer
#* 2 μL 10X FastDigest Buffer
#* 2 '''ug''' pLentiCRISPRv2 backbone plasmid
#* 2 '''ug''' pLentiCRISPRv2 backbone plasmid
Line 39: Line 40:
# Spin tube down for few seconds
# Spin tube down for few seconds
# Digest by incubating in waterbath at 37°C for 90 minutes
# Digest by incubating in waterbath at 37°C for 90 minutes
# Measure concentration on Nanodrop


'''Dephosphorylate:'''
'''Dephosphorylate:'''
Line 49: Line 49:
# Incubate in waterbath at 37°C for 1 hour
# Incubate in waterbath at 37°C for 1 hour
# Stop reaction by incubating at 75°C for 5 minutes.
# Stop reaction by incubating at 75°C for 5 minutes.
# Measure concentration on Nanodrop


'''Anneal oligos:'''
'''Anneal oligos:'''
# Prepare the following:
# Prepare the following:
#* Dilute primers to 100 μM in new tubes  
#* Dilute primers to 100 μM (100pmol/uL) in new tubes  
#* The negative control, using H20 instead of anneal oligo's.
# Add the following in a PCR tube:
# Add the following in a PCR tube:
#* 6.5 μL ddH20  
#* 6.5 μL ddH20  
#* 1 μL oligo 1 (100 μM)
#* 1 μL oligo 1 (100 μM)
#* 1 μL oligo 2 (100 μM)
#* 1 μL oligo 2 (100 μM)
#* 10X T4 Ligation Buffer
#* 1 μL 10X T4 DNA Ligation Buffer<br />'''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 T4 PNK
#* 0.5 μL T4 PNK
# Place in thermal cycler and run using the following program:
# Place in thermal cycler and run using the following program:
#* Step 1: 37°C for 30 min
#* Step 1: 37°C for 30 min
#* Step 2: 95°C for 5 min
#* Step 2: 95°C for 5 min
#* Step 3: Decrease 5°C every minute until at 25°C
# Remove tubes and let tubes sit at room temperature for 50 minutes.
# Briefly spin tubes to draw all moisture from the lid
# 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.
# Dilute annealed oligos at a 1:200 dilution into sterile water.
# Keep on ice or 4°C


'''Ligate annealed oligo into backbone:'''
'''Ligate annealed oligo into backbone:'''
Line 83: Line 85:


'''Storage / next steps:'''
'''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.
* Then either store at -20°C or move on to [[Mike_Barnkob:Protocols/CRISPR/Transforming_bacteria|transform construct 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===

Latest revision as of 03:14, 16 February 2015

← Front page

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:

Retrieved from "https://openwetware.org/mediawiki/index.php?title=Mike_Barnkob:Protocols/CRISPR/Cloning_oligos_into_backbone&oldid=864718"