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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) 10μM F/R primer mix AmpliTaq Gold 360 Master Mix Water Total
Stage Temp Time Cycles
Enzyme activation 10 min Denature 30 sec Anneal 30 sec
Extend 30 sec
Final extension 7 min 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 5 min
Annealing 95-85°C
Hold 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 T7 endonuclease I (M0302) OR water dx.doi.org/10.17504/protocols.io.g93bz8n

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