Hello
i am doing an experiment knock out a gene in epithelioma papulosum cyprini (EPC) cell using precision cas9 smartnuclease vector system from system bioscience. following the guidelines, i transfected the cell and select the positives ones. i extracted the genomic DNA from the control cell and transfected cell. i did PCR to amplify the target region.
after that i used the T7 nuclease assay from NEB to confirm the mutation caused by CRISP CAS9, but the positive control was cut also as you can see in the attached picture.
the T7 nuclease protocol was done as mentioned in the NEB guideline.
i want to know why the control digest?
and if there is a different protocol for T7 nuclease to use it will be helpful for me.
thanks.
We also experienced similar trouble with the similar Surveyor assay.... Anyway, have you considered simply looking in Western Blot for KO?
If you use a donor DNA, you could try to detect the junction between the donor and genomic DNA.
If you do not use donor DNA, PCR of the deletion site, cloning and sequencing of 10 to 20 cloned DNA.
Thank you for answering my question.
Mr.Stéphane Roche
I am not using donor vector so i will clone the target region and submit for sequencing.
after confirmation of the mutation i would like to try the T7 nuclease assay to confirm the mutation. so if possible someone can give me a protocol which worked fine using T7 nuclease.
Mr.Christian Gabriel
i will do western blot to confirm the KO of the gene but i like to confirm first the mutation.
thanks for your help.
Did you try to clone the pcr amplified fragment ( from target genomic DNA) in to a T-easy system ? that way you can have a closer look at your target site and you can also have an estimate about the efficiency of your editing
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