T7 Endonuclease I (T7E1) Assay Protocol for CRISPR/Cas9 gRNA validation
Genomic DNA primer design1   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:
|10μM F/R primer mix||1μl|
|AmpliTaq Gold 360 Master Mix||25μl|
9   Run PCR reaction with the following conditions:
|Enzyme activation||95°C||10 min||1X|
|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
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.