gRNA_Cloning Vector

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	For goldengate reaction, there is a low and a high concentration mixture available for T4 ligase. For single inserts, the low concentration is just fine, but the follow-up article on golden gate cloning (PMID: 19436741) found that the high-concentration T4 was better for multiple insert cloning.

Protocol tips
For goldengate reaction, there is a low and a high concentration mixture available for T4 ligase. For single inserts, the low concentration is just fine, but the follow-up article on golden gate cloning (PMID: 19436741) found that the high-concentration T4 was better for multiple insert cloning.

Publication protocol

Generation of RMA.Trh4 Db or Kb knock-out cells using CRISPR/Cas9 system
CRISPR/CAS9 sgRNA's targeting both Db and Kb were designed using online CRISPR Design software (http://crispr.mit.edu). The sgRNA sequence (5′- AGATGTACCGGGGCTCCTCG-3′) was cloned into a sgRNA expression vector (Addgene 41824) using a Gibson In-fusion kit. RMA-Trh4 cells were transfected with the vector containing the sgRNA and a plasmid containing Cas9 WT (Addgene 41815), using lipofectamine 2000. Flow cytometry analysis of cells transfected with the sgRNA/CAS9WT plasmids generated both Db and Kb deficient cell populations, in line with homology between the genes. From these transfected cells, Db or Kb –deficient cells were FACS-sorted and used for further experiments.



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Manufacturer protocol

Download the product protocol from Addgene for gRNA_Cloning Vector below.

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