T7 Endonuclease I (T7E1) Assay Protocol for CRISPR/Cas9 gRNA validation
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.
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.
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 |
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.
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 85-25°C |
-2°C/sec -0.1°C/sec |
|
| 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
AAV(Adeno-Associated Virus) vector system
AAV Rep-Cap plasmids (serotypes-specific AAV RC plasmids)
Facebook
LinkedIn
Twitter
