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

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(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])
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* 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)===


'''Harvest spleen:'''
'''Digest backbone:'''
# Euthanize mouse by cervical dislocation.
# Prepare the following:
# Dissect out spleen<br />'''Note:''' Dip whole mouse into pipet-tip holder with 70% ethanol in it.<br />'''Note:''' Keep hair out of incision.  
#* 100mM DTT: 1 μL 1M DTT into 9 μL ddH20.
# Using forceps, quickly dip spleen into 70% ethanol.
# Add the following in order to Eppendorf tube:
# Transfer spleen into flask with media, keep on ice.
#* 15.5 μL dH20
# In tissue culture hood, cut spleen into smaller pieces (2-4 in total), around 20-30mg per piece.
#* 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


'''Homogenize tissue and lyse red blood cells:'''
'''Dephosphorylate:'''
# Work in tissue culture hood
# Add the following to the digested backbone:
# Add 1mL of R10 to 50 ml Falcon tube. Place 70μM cell strainer on tube and add part of spleen on top of this. <br />'''Note:''' The mRNA isolation kit can maximum handle 30 mg of tissue. Aim for 10-20 mg per part.<br />'''Note:''' Left-over spleen can be kept in RNALater media from Qiagen.
#* 2 μL 10x FastAP buffer
# Take out plunger of 2.5ml syringe, and mesh spleen through strainer with blunt end of the plunger.<br />'''Note:''' Flush cells through with a little media afterwards.<br />'''Note:''' When done, add up to 5 ml R10.
#* 1 μL FastAP (1 units)
# Spin cells down at 1500 RPM for 5 min and remove supernatant.
# Gently mix by pipetting
# Resuspend in 2 ml of red blood cell lysis buffer and leave on ice for 5 min.
# Spin tube down briefly
# Add 5 ml of media, spin cells down at 1500 RPM for 5 min and remove supernatant.
# Incubate in waterbath at 37°C for 1 hour
# '''Optional:''' Resuspend in 1 ml of media and count cells, otherwise continue to mRNA isolation.
# Stop reaction by incubating at 75°C for 5 minutes.


'''mRNA isolation:'''
'''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.


''Follows Qiagen RNeasy Mini Kit instructions and presumes all reagents have been prepared.''
'''Ligate annealed oligo into backbone:'''
# (Spin cells down at 1500 RPM for 5 min and remove supernatant)
# Mix the following together in 1.5 Eppendorf tube:
# Disrupt and homogenize:
#* '''50ng''' digested plasmid backbone 
#* Resuspend in 600μL buffer RLT and pipet up and down several times.
#* 1 μL of diluted, annealed oligos
#* Transfer to 1.5 Eppendorf tube
#* 5 μL 2X Quick Ligase Buffer
#* Centrifuge the lysate for 3 min at full speed and transfer the supernatant to new 1.5 Eppendorf tube.<br />'''Note:''' The lysate is normally between 250-600 μL.<br />'''Note:''' When adding RLT, liquid can become viscus, so pipet slowly.
#* Add ddH20 up to 10 μL
#* Add 1 volume of 70% ethanol to the lysate, and mix immediately by pipetting (don't vortex).
#* 1 μL Quick Ligase
# Transfer 700 μL of sample to an RNeasy spin column placed in a 2 ml collection tube. Close  lid gently, and centrifuge for 15 s at 10,000 rpm. Discard the flow-through.
# Mix gently by pipetting
# Add 700 μL Buffer RW1 to the RNeasy spin column. Close lid gently, and centrifuge for 15 s at 10,000 rpm. Discard the flow-through.
# Centrifuge briefly for a few seconds
# Add 500 μL Buffer RPE to the RNeasy spin column. Close lid gently, and centrifuge for 15 s at 10,000 rpm. Discard the flow-through.
# Ligate: let reaction sit for 10 min at room temperature.
# Add 500 μl Buffer RPE to the RNeasy spin column. Close lid gently, and centrifuge for 2 minutes at 10,000 rpm. Discard the flow-through.
# Chill on ice
# Place the RNeasy spin column in a new 1.5 ml collection tube. Add 30–50 μL RNase-free water directly to the spin column membrane. Close the lid gently, and centrifuge for 1 min at 10,000 rpm to elute the RNA.
# Label and store at -20°C.


'''Quality control:'''<br />
'''Quality control:'''
''Using a NanoDrop''
# Check product by running 1 μL on gel. If many bands are showing up consider gel purification.
# A260/A280: ratio should be close to 1.8-2.0 [http://en.wikipedia.org/wiki/Nucleic_acid_quantitation].
# A260/A230: ratio should be close to 2.0 when isolating low amounts of RNA. Values < 1.0 indicates contamination with salts or rests of phenol or protein in the RNA solution.
''Gel:''
# Make [[Mike_Barnkob:Protocols/Cloning/Gels|1% agarose gel]]
# Mix in Eppendorf tube:
#* 4 μL dH20
#* 5 μL Blue Loading Dye (6X)
#* 1 μL RNA sample
# Run the gel at 100 V until the fastest dye has moved 2/3 of the gel length
# Visualize gel using a UV transilluminator. A good extraction should have two clearly distinguishable bands, like below:
[[Image:Mike_Barnkob_2015-01-22_mRNA_gel.jpg]]


'''Storage:'''
'''Storage / next steps:'''
* Purified RNA may be stored at –20°C or –70°C in RNase-free water. Under these conditions, no degradation of RNA is detectable after 1 year.
# 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===


* RNeasy Mini Handbook, p37: http://www.qiagen.com/gb/resources/resourcedetail?id=14e7cf6e-521a-4cf7-8cbc-bf9f6fa33e24&lang=en
pLentiCRISPRv2 protocol:
* RNA quality control: http://biomedicalgenomics.org/RNA_quality_control.html
* Addgene: https://www.addgene.org/static/data/plasmids/52/52961/52961-attachment_B3xTwla0bkYD.pdf
* RNA qualitl control using gel electrophoresis: http://www.flychip.org.uk/protocols/gene_expression/rna_qc.php
 
* Bürgmann et al, 2003: aem.asm.org/content/69/4/1928.full
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

← 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

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:

Protocol 1 (Yale style)

Digest backbone:

  1. Prepare the following:
    • 100mM DTT: 1 μL 1M DTT into 9 μL ddH20.
  2. 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
  3. Gently mix by pipetting
  4. Spin tube down for few seconds
  5. Digest by incubating in waterbath at 37°C for 90 minutes
  6. Measure concentration on Nanodrop

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.

Anneal oligos:

  1. Prepare the following:
    • Dilute primers to 100 μM in new tubes
  2. 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
  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
    • Step 3: Decrease 5°C every minute until at 25°C
  4. Briefly spin tubes to draw all moisture from the lid
  5. Put on ice or 4°C
  6. Dilute annealed oligos at a 1:200 dilution into sterile water.

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:

  1. 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:

  1. 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
  2. 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
  3. Dilute annealed oligos at a 1:200 dilution into sterile water.

Digest backbone and dephosphorylate

  1. 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
  2. Gently mix by pipetting
  3. Spin tube down briefly
  4. Digest by incubating in waterbath at 37°C for 10 minutes
  5. Inactivate enzymes by incubating at 80°C for 20 minutes

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:

  1. 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:

Annealing oligos:

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