I performed this kit upon my cells (which were transfected with LentiCrispr v2), purified those with Roche PCR purification kit, and it did not work and I only got 1 band after Surveyor digestion (Ref: Genome engineering using the CRISPR-Cas9 system, F. A. Ran, F. Zhang.).
I decided to perform it with only controls (G and C), same as recommended. In both applications, I observed PCR products (with Herculase II PCR buffer) but I observed same result (only 1 band) after Surveyor digestion again.
Ta was 65 Celcius,
Program for generating PCR products and performing heteroduplex formation were applied same as in the user guide,
Samples were run on 2% agarose gel,
SYBR safe was used,
Samples were loaded with 6X loading buffer (5µL samples+1µL loading buffer)
Images were collected with Chemi-doc Bio-Rad.
Then, I tried with;
1. I prepared heteroduplex formation with using Herculase,
2. Since Herculase is not compatible with Surveyor Kit (not in the list of user guide), I also prepared PCR products of controls (G and C) with GoTaq PCR buffer and polymerase too. (However, I did not get any PCR products from GoTaq application),
3. Besides, after generating PCR products, I prepared heteroduplex formation both with using Herculase and GoTaq PCR buffers,
4. In addition, I used Surveyor Enhancer S and Surveyor Nuclease S from new tubes (which were supplied with the kit),
After all these applications, I only observed 1 band when I treated samples with Surveyor Nuclease S.
So, if someone can help me with finding a solution, I will gladly accept it and appreciate that.
Thank you..
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