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

DNA:

• Backbone plasmid
• Guide oligo's

Enzymes and reagents:

• FastDigest BsmBI (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)
• ddH20

Transformation:

• Stbl3 bacteria

Protocol

Harvest spleen:

1. Euthanize mouse by cervical dislocation.
2. Dissect out spleen
   Note: Dip whole mouse into pipet-tip holder with 70% ethanol in it.
   Note: Keep hair out of incision.
3. Using forceps, quickly dip spleen into 70% ethanol.
4. Transfer spleen into flask with media, keep on ice.
5. In tissue culture hood, cut spleen into smaller pieces (2-4 in total), around 20-30mg per piece.

Homogenize tissue and lyse red blood cells:

1. Work in tissue culture hood
2. Add 1mL of R10 to 50 ml Falcon tube. Place 70μM cell strainer on tube and add part of spleen on top of this.
   Note: The mRNA isolation kit can maximum handle 30 mg of tissue. Aim for 10-20 mg per part.
   Note: Left-over spleen can be kept in RNALater media from Qiagen.
3. Take out plunger of 2.5ml syringe, and mesh spleen through strainer with blunt end of the plunger.
   Note: Flush cells through with a little media afterwards.
   Note: When done, add up to 5 ml R10.
4. Spin cells down at 1500 RPM for 5 min and remove supernatant.
5. Resuspend in 2 ml of red blood cell lysis buffer and leave on ice for 5 min.
6. Add 5 ml of media, spin cells down at 1500 RPM for 5 min and remove supernatant.
7. Optional: Resuspend in 1 ml of media and count cells, otherwise continue to mRNA isolation.

mRNA isolation:

Follows Qiagen RNeasy Mini Kit instructions and presumes all reagents have been prepared.

1. (Spin cells down at 1500 RPM for 5 min and remove supernatant)
2. Disrupt and homogenize:
   • Resuspend in 600μL buffer RLT and pipet up and down several times.
   • Transfer to 1.5 Eppendorf tube
   • Centrifuge the lysate for 3 min at full speed and transfer the supernatant to new 1.5 Eppendorf tube.
     Note: The lysate is normally between 250-600 μL.
     Note: When adding RLT, liquid can become viscus, so pipet slowly.
   • Add 1 volume of 70% ethanol to the lysate, and mix immediately by pipetting (don't vortex).
3. 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.
4. 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.
5. 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.
6. 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.
7. 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.
8. Label and store at -20°C.

Quality control:
Using a NanoDrop

1. A260/A280: ratio should be close to 1.8-2.0 [1].
2. 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:

1. Make 1% agarose gel
2. Mix in Eppendorf tube:
   • 4 μL dH20
   • 5 μL Blue Loading Dye (6X)
   • 1 μL RNA sample
3. Run the gel at 100 V until the fastest dye has moved 2/3 of the gel length
4. Visualize gel using a UV transilluminator. A good extraction should have two clearly distinguishable bands, like below:

Storage:

• 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.

References

• RNeasy Mini Handbook, p37: http://www.qiagen.com/gb/resources/resourcedetail?id=14e7cf6e-521a-4cf7-8cbc-bf9f6fa33e24&lang=en
• RNA quality control: http://biomedicalgenomics.org/RNA_quality_control.html
• 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