TITLE: Target Guide Sequence Cloning Protocol Version 2
AUTHORS: Skye Waterland, Yang Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Create single gRNA vectors for targeted cloning utilizing CRISPR or CRISPR-based systems. </div></div>

[STEPS]
?. [Lentiviral vector digestion]
Digest and dephosphorylate
?. [Lentiviral vector digestion]
Add  to a .
40 µl
1.5 mL
?. [Lentiviral vector digestion]
Add  of  to solution.
5 µl
?. [Lentiviral vector digestion]
Add  of to solution.
3 µl
*Note: we used BsmBI Cat #R0580, NEB, but this one has been discontinued. #R0739L is considered as an effective replacement.*
?. [Lentiviral vector digestion]
Add  of  to solution.
2 µl
?. [Lentiviral vector digestion]
Close cap on microcentrifuge tube and place in  for  on until all of the solution is at the bottom of the tube.
?. [Lentiviral vector digestion]
Place microcentrifuge tube with vector digestion mixture in a on
55 °C
?. [Lentiviral vector digestion]
Close lid and set a timer for
?. [Gel purify the digested plasmid from Step 1 ]
Prepare gel
?. [Gel purify the digested plasmid from Step 1 ]
Create gel concentration of 1.2-1.5%for  of solution.
100 mL
?. [Preparing the gRNAs]
Design gRNA sequence for CRISPR strategy using CRISPR direct.
?. [Preparing the gRNAs]
Order oligos from Sigma-Aldrich (RRID:SCR_008988).
?. [Preparing the gRNAs]
Create a dilution from stock oligos at a 1:10 ratio in .
Diluted oligo concentration should be  .
?. [Phosphorylate and anneal each pair of oligos]
Prepare phosphorylation/annealing reaction
?. [Phosphorylate and anneal each pair of oligos]
Add  each of  Oligo 1 (F),  Oligo 2 (R), and
1 µl
?. [Phosphorylate and anneal each pair of oligos]
Add
0.5 µl
?. [Phosphorylate and anneal each pair of oligos]
Vortex and microcentrifuge
?. [Setting up and incubating the ligation reaction ]
Making the ligation reaction
?. [Setting up and incubating the ligation reaction ]
Add
2.2 µl
?. [Setting up and incubating the ligation reaction ]
Add  each of , diluted oligo duplex from , and .
1 µl
?. [Setting up and incubating the ligation reaction ]
Lightly vortex and microcentrifuge
?. [Transformation into E. coli bacteria ]
Prepare LB Agar plates
?. [Picking Colony for Suspension Growth ]
Picking colony
?. [Picking Colony for Suspension Growth ]
Culture the bacteria
?. [Run PCR to Identify Positive Clones ]
Prepare Master PCR Mix by adding each of the below reagents to a microcentrifuge tube.
Before you start: multiply each volume below by the same amount (x 10, 15, etc.) according to how much master mix you need to run your sets.
?. [Run PCR to Identify Positive Clones ]
15.875 µl
?. [Run PCR to Identify Positive Clones ]
Loading and Prepping PCR wells
?. [Run PCR to Identify Positive Clones ]
Place PCR reaction in thermocycler and run PCR.
Settings should be as follows:1) 95C for 5mins (ramp up). 2) 95C for 30sec. 3) 55C for 45 sec.  4) 72C for 45 sec.  - x35 cycles steps 2-4. 5) 72 for 10mins.
?. [Running Gel for PCR Product Verification]
Prepare a gel of 2% concentration. Run gel and examine bands. Desired band length is about 200bp with the gRNA insertion.
?. [Congrats! ]
You have successfully transformed a lentiviral vector with your gRNA sequence of interest! For confirmation, feel free to sequence your vector.
?. [Gel purify the digested plasmid from Step 1 ]
Run gel
?. [Gel purify the digested plasmid from Step 1 ]
Isolate 2kb and 8kb band. Collect 8kb (8318 bp) band for gel purification.
?. [Phosphorylate and anneal each pair of oligos]
Addto a microcentrifuge tube.
6.5 µl
?. [Phosphorylate and anneal each pair of oligos]
Place the phosphorylation/annealing reaction in a
Settings should be as follows:   for  ,  for  , and then ramp down to   at  / .
?. [Setting up and incubating the ligation reaction ]
Place  of  in a microcentrifuge tube.
4.8 µl
?. [Setting up and incubating the ligation reaction ]
Incubate the ligation reaction at room temperature for-
?. [Transformation into E. coli bacteria ]
Wipe down your bench with at least 70%and light a bunsen burner.
?. [Transformation into E. coli bacteria ]
Remove Agar Ampicillin Plates  from  and let warm up to room temperature.
250 µl
4 °C
?. [Transformation into E. coli bacteria ]
E. coli competent cell transfection
?. [Transformation into E. coli bacteria ]
Take competent cells out of  and thaw on ice (- ).
-80 °C
?. [Transformation into E. coli bacteria ]
Add  of E.coli cells to  of DNA in a microcentrifuge tube next to the bunsen burner.
100 µl
10 µl
When working with the E. coli, be very diligent and make sure you are working in the sterile area of your bench near the bunsen burner flame.
?. [Transformation into E. coli bacteria ]
Gently flick tube a few times with your finger to mix.
?. [Transformation into E. coli bacteria ]
Incubate the competent cell/DNA mixture on ice for .
?. [Picking Colony for Suspension Growth ]
Pick 10-15 colonies from each agar plate to suspend in an LB solution.
We originally selected only 4 colonies from each plate, but did not have a successful PCR. To increase chances of successfully amplifying the plasmid vector, we suggest picking 10-15 colonies.
?. [Picking Colony for Suspension Growth ]
Place each colony in a tube with of LB.
3 mL
?. [Picking Colony for Suspension Growth ]
Place tubes into shaking incubator at  overnight.
37 °C
?. [Picking Colony for Suspension Growth ]
Before you get started
Start this whole section (picking colony and suspension growth) sometime in the late afternoon (~4-5pm), so that you can run PCR for the bacterial plasmids you collect the next morning (~16 hours later). More than 24 hours of incubation can cause other non-ampicillin resistant bacteria to grow in the suspension tubes.
Set up an aseptic area for handling the bacterial colonies.
?. [Run PCR to Identify Positive Clones ]
2.5 µl
?. [Run PCR to Identify Positive Clones ]
0.5 µl
?. [Run PCR to Identify Positive Clones ]
Forward Primer hU6-02
2 µl
Forward Primer hU6-02 sequence: TAATTAGAATTAATTTGACTOrdered from Sigma-Aldrich (RRID:SCR_008988).
?. [Run PCR to Identify Positive Clones ]
Reverse Primer (gRNA reverse primer oligo)
2 µl
?. [Run PCR to Identify Positive Clones ]
0.125 µl
?. [Run PCR to Identify Positive Clones ]
Aliquot equal proportions of master mix to each PCR well (we used ).
23 µl
?. [Run PCR to Identify Positive Clones ]
Add  of each bacterial suspension sample from   to each PCR well.
2 µl
Total volume of Master Mix and Sample should be   in each well.
?. [Run PCR to Identify Positive Clones ]
Seal off PCR wells tightly with a clear plastic cover or tube tops.
?. [Run PCR to Identify Positive Clones ]
Centrifuge  .
Centrifuge: 1200 33, 1 min
?. [Transformation into E. coli bacteria ]
Place transformation tube(s) into water bath at  for  - to heat shock E. coli cells.
42 °C
?. [Transformation into E. coli bacteria ]
Place the transformation tube(s) back on ice for .
?. [Transformation into E. coli bacteria ]
Add  (without antibiotic) or to the tube(s).
250 µl
?. [Transformation into E. coli bacteria ]
Place tube(s) in  shaking incubator for -.
37 °C
?. [Transformation into E. coli bacteria ]
Plate all of transformation onto LB agar plate(s) with ampicillin. Incubate plates at  overnight.
37 °C