03 November 2021 1 2K Report

The cell line I planned to do CRISPR KI was already tagged by GFP. Two genes are going to be sequentially knocked in with mcherry and mBFP. So I planned to use pcDNA 3.1 for donor DNA and lenti CRISPR V2 for gRNA delivery, both without fluorophores. Homology arms in pcDNA 3.1 are going to be about 700bp long each, adding together, the KI fragment is going to be about 2000bp. Since V2 has puromycin resistance gene, I could first do puro screening for 2-3 days, and then go on with single clone cultivation and sequencing. Please give me some comments and suggestions for my experimental design. I'll be grateful for any input since I must have overlooked lots of important details. Thanks in advance.

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