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Genomic DNA primer design

1   Design genomic DNA primers that are approximatley 18 to 22 basepairs in length and have 45-55% GC content. For best results, use primers with a Tm greater than 55 C. Design primers to yield amplicon length between 400-500 bp. In addition, design primers so that the predicted cleavage site is not in the center of the amplicon and the detection reaction will yield two distinct product bands.

DNA extraction

2   Spin down cells transfected with CRISPR constructs at 200g for 5 min at 4°C.

3   Carefully remove supernatant and wash once with 1XPBS.

4   Extract genomic DNA using Epicentre QuickExtract.

5   Dilute samples to 40ng/μl.

Amplification with AmliTaqGold 360 Master Mix (Applied Biosystems 4398881).

6   Vortex DNA.

7   Set up the following components for PCR:
Component Sample
gDNA (40ng/μl) 2.5μl
10μM F/R primer mix 1μl
AmpliTaq Gold 360 Master Mix 25μl
Water 21.5μl
Total 50μl

8   For best results, add 5 μl of 360 GC Enhancer per 50μl PCR reaction when amplifying GC rich loci.

9   Run PCR reaction with the following conditions:
Stage Temp Time Cycles
Enzyme activation 95°C 10 min 1X
Denature 95°C 30 sec 40X
Anneal 55°C(Tm) 30 sec
Extend 72°C 30 sec
Final extension 72°C 7 min 1X

10   Clean up PCR reaction (Qiagen MiniElute PCR purification Kit). If ~50 ng is present, run 3 μl of PCR product on a 1.5-2% agarose gel. If a single band of expected size is present, proceed to the denaturing and annealing step.

Hybridization of PCR products

11   In a PCR tube put the following:
Make two tubes for each DNA sample - one where T7 Endonuclease I is added and one with no T7E1 enzyme.
200 ng DNA
2 uL 10X NEB buffer 2
To19 μl Nuclease-free Water

12   Hybridation conditions
Step Temperature Ramp Rate Time
Denaturation 95°C 5 min
Annealing 95-85°C
Hold 4°C Hold

13   Add T7 Endonuclease I to one tube for each DNA sample. (Add water to the other tube.)
Component 20 μl reaction
Annealed PCR product 19 μl
T7 endonuclease I (M0302) OR water 1 μl

14   Incubate for 15 minutes at 37 C.

15   Run products on 2% agarose gel or 10-20% TBE polyacrylamide gel and a 1X TBE buffer.
If the sgRNA created indels in your cells, you should see two small fragments below the wildtype band of 500 bp.

Ref: dx.doi.org/10.17504/protocols.io.g93bz8n