Dear all,
I try to detect possible mutations within my DNA.
For this, I use the Surveyor Nuclease S Assay (Cel-I-Assay).
After heteroduplex Formation andTreatment with Surveyor Nuclease, I don´t get any product.
Strangely, I checked my DNA after PCR purification via gel electrophoresis and detected a clear and distinct band, as expected.
So, what happended to my DNA? Any suggestions?
Best regards
did the control samples included in the kit work and are you seeing your size standard and the uncut dna control on your gel. Are other workers getting pictures of their checker gels from the transilluminator system that you are using to photograph your results?
Dear Paul,
yes, the controls work. I additionaly tested different nuclease aliquots, but there seems to be no degradation of the enzyme...I also checked the product after the heteroduplex Formation...mysterious!
Hi Bastian,
I am assuming that you have an uncut heteroduplex band in your nuclease gel track. The 2 most likely reasons are that the heteroduplex is , in fact , a homoduplex so cannot cut. This will happen if there are no mutations/snps. Alternatively if you have normal template contamination in your pcr mix then what you expect to be mutated pcr product is,in fact, normal. What do the negative controls of you PCR look like?
I am attaching an iDT guide to surveyor just in case there is something useful concerning the surveyor buffer and various pcr buffers ( page 22) which may be of interest.regards
paul
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