MLM3636

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	There is no need to perform a negative control golden-gate reaction (without insert) as it will always contain colonies so not a good indicator of cloning success.

Protocol tips
There is no need to perform a negative control golden-gate reaction (without insert) as it will always contain colonies so not a good indicator of cloning success.

Publication protocol

Generation of gRNA Expression Vectors
The SpCas9 plasmid JDS246 (Plasmid 43861) and the gRNA expression plasmid MLM3636 (Plasmid 43860) were obtained from a non-profit plasmid share repository (Addgene, Cambridge, MA, USA). Suitable CRISPR target sites within Prnp Exon 3 positive and negative strands were identified using the ‘CRISPR Design Tool’ (http://crispr.mit.edu/) described in the Results section. The respective oligonucleotide pairs were obtained from Life Technologies (Burlington, ON, Canada) and were customized to include overhangs compatible for ligation into MLM3636 linearized by digestion with BsmB1 (cat. no. R0580S; New England BioLabs, Ipswich, MA, USA), a cloning site located in this vector on the 3′ side of a U6 promoter element. Oligonucleotides were phosphorylated with polynucleotide kinase (cat. no. EK0031; Fermentas, Ottawa, ON, Canada), annealed and inserted into the gRNA plasmid using T4 DNA ligase (cat no. M0202S; New England Biolabs) and transformed into Turbo competent E. coli (cat no. C2984H; New England Biolabs).

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Reviews

Discussion

Papers

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Manufacturer protocol

Download the product protocol from Addgene for MLM3636 below.

Download manufacturer protocol

Videos

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