![]() I guess T4 ligase incubate time is a key factor, but maybe different sgRNA also vary the correct rate, fortunately I get all colonies I want, for those many times try, I pick 4 key factor :1, cut your vector thoroughly ,cut for a longer time maybe better 2, linear vector form gel extraction must to be a good quality I means have a proportion concentration and have good rate of 260/280=1.8-2.0 and 260/230=1.5-2.0(impurity vector may harm the T4 ligase efficiency ) 3.ligand your annealed oligos and linear vector for longer time maybe better 3,Choose competent cell with recombination gene (like Rec.)deletion or mutation to avoid recombination, in this protocol stbl3 is a good choice but top10 is not OK. Command Line: Creating Maps of DNA Sequences. 1) In a thermocycler, digest the vector with Esp3I (Thermo Fisher, add DTT to 1mM) at 37C for 3-4 hours, inactivate at 65C for 20 mins, keep at 4C. ![]() Use with SnapGene software or the free Viewer to visualize additional data and align other sequences. ![]() SnapGene File: Plasmid sequence and SnapGene enhanced annotations. Use text editor or plasmid mapping software to view sequence. GenBank File: Plasmid sequence and annotations. To set the number of fragments for ligation, click the ' + ' or ' ' buttons. Image: Illustrated plasmid map in PNG format. the complex network of host interaction of the human cytomegalovirus (hcmv) involves the regulatory protein kinase pul97, which represents a viral cyclin-dependent kinase (cdk) ortholog. Dear Mohamad, I get my sequence result this morning, I design six sgRNA and insert them into lenticrispr v2 respectively, even though I use the quickly T4 DNA ligase which ligate at room temperature for 5min, last time I just get 20% correct rate,but this time ligation at 22℃ for 2h the correct rate range from 25%-50%. By default the Linear Ligation tool starts with two fragments. To examine whether YAP-positive puncta occur independently of IF, we engineered TDCLs in which YAP is labeled endogenously with monomeric enhanced green fluorescent protein (mEGFP) using CRISPR-Cas9 genome editing ( Figures S2 B and S2C).
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