Hi all,
I have been optimizing Surveyor nuclease assay to detect indels in the genomic DNA of Cas9 transfected HEK cells. To begin with, I am using 300 ng and 400 ng of total DNA (Control DNA procided with the kit) in separate reactions with 0.5 uL of nuclease (half of the recommended since I obtained smear using 1 uL of nuclease) and 20 mins incubation at 42 degrees. I can see the original PCR product now as opposed to before, when I had used 1 uL of the nuclease. However, I still cannot see the fragments and observe a smear in both test (heteroduplex) and control (homoduplex). Can anyone suggest how do I overcome this problem?
Hey,
I had similar problems and also tried to optimize it for weeks. Then I decided to switch to the T7 Assay, since it's cheaper, faster and giving good results. You should also think about this! Do you have single nucleotid changes or larger indels? In case of indels the T7 assay is even more sensitive!
Good luck!
Hi Ariane,
Did you also try the Surveyor from Integrated DNA Tech?
Hey,
yes I did! But it was never really working in my hands, I couldn't see clear bands, only smear or nothing at all!
Hi Ariane,
I followed the optimization chart given in the Surveyor manual from IDT and it worked for me! Anyway, thank you for your help!
Hi Kathleen,
I used Optimase polymerase from Transgenomic (now ADS Biotech). Also, I used Surveyor from Integrated DNA technologies. They have a manual which provides different volumes of DNA (PCR product) to be added along with proportionate amounts of MgCl2 and equal volumes of Nuclease and Enhancer in all of them. To amplify, I used one template in duplicates and then combined them after PCR for PCR cleanup (QIAquick PCR cleanup kit). For elution after PCR cleanup, I used 30 uL of elution buffer which gave me a good yield anything between 40-80 ng/uL. For concentrations more than 50 ng/uL, I made up the concentration upto 50 ng/uL with elution buffer used in PCR cleanup kit. I followed the table given in the manual with Control G and Control C plasmids given in the Surveyor kit. This helped me get the optimum volume:volume ratio of DNA:MgCl2:Surveyor:Enhancer.
Hope this was helpful!
Hi Kathleen,
Lately, I am having little trouble in getting crisp cleavage products after Surveyor treatment in controls. One possible reason could be I am using DNA which is more than a month old. I am not sure about this reason but you might want to take this into account. Alternatively, there is another similar nuclease T7E1 available from NEB which you can try as well. I am not sure if you have to optimizae that assay as well but you can try both of them in single experiment and see which one works better.
Were you able to improve DNA quality by changing PCR volume, MgCl2, nuclease and enhancer? I am having the same problem. Do you still get a smear eve when you purify the DNA? I was wondering if this has something to do with incompatibility with buffers.
Hi Amanda, I followed the table given in the manual of the Surveyor Assay from Integrated DNA Technologies. I cleaned up PCR products of Control G and C and diluted them to 50 ng/uL. I used this stock to then set up the reactions as given in that table. This worked out well for me and I chose 600 ng of total DNA to be good for my subsequent experiments. Hope this works!
hey,
I use 400 ng DNA product and 1 ul of the T7, but when I run Gei, I could see nothing at all, who can tell me how the do with this problem?
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