Hello,
I am trying to do knock-out in cell cultures using CRISPR.
The cells are being transfected with a plasmid containing the essentials for CRISPR and a GFP marker. The transfection reates are quite low: about 20%, and out of those probably not all have been edited successfuly with CRISPR. After that DNA is extracted. Before I do FACS sorting for GFP and sequencing for detection of mutations/insertions/deletions, I wanted to just get an answer YES/NO if the CRISPR worked (don't care exactly what happened in the gene), a bit like pooling. So I thought to do HRM analysis to see if there is a difference between transfected cells and non transfected cells. The question is if it is possible to detect in HRM mutations even though not all cells and therefore not all the DNA is actually different from the control (non transfected cells).
Bottom line, is it possible to detect differences in HRM if only about 10% of the template is edited/different between the samples.
Thank you
I am trying to do knock-out in cell cultures using CRISPR.
The cells are being transfected with a plasmid containing the essentials for CRISPR and a GFP marker. The transfection reates are quite low: about 20%, and out of those probably not all have been edited successfuly with CRISPR. After that DNA is extracted. Before I do FACS sorting for GFP and sequencing for detection of mutations/insertions/deletions, I wanted to just get an answer YES/NO if the CRISPR worked (don't care exactly what happened in the gene), a bit like pooling. So I thought to do HRM analysis to see if there is a difference between transfected cells and non transfected cells. The question is if it is possible to detect in HRM mutations even though not all cells and therefore not all the DNA is actually different from the control (non transfected cells).
Bottom line, is it possible to detect differences in HRM if only about 10% of the template is edited/different between the samples.
Thank you
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