NEB has developed a protocol to quickly and easily change the targeting of a single-guide RNA in a sgRNA plasmid or to create an sgRNA plasmid library. The method uses a single DNA oligonucleotide, a restriction enzyme digested plasmid and the NEBuilder HiFi DNA Assembly Master Mix.
Choose a target sequence or target sequence using a design tool of your choice. Design a single-stranded DNA oligo, containing a target sequence flanked by a partial promoter sequence and scaffold RNA sequence. In this animation the U6 promoter is used.
Prepare the DNA oligo. Assemble the reaction mix by combining DNA oligo, restriction-enzyme linearized plasmid and water.
Add NEBuilder HiFi DNA Assembly Master Mix and incubate for 1 hour at 50 degrees Celsius.
Transform NEB 10-beta Competent E.coli with the assembled product.
Spread outgrowth on plate with antibiotic and incubate overnight at 37 degrees Celsius.
Pick colonies to grow, and purify the plasmid DNA for sequencing.
Some traditional methods require synthesis, phopsphorylation, annealing, and ligation of two oligos into a digested and dephosphorylated vector. This new protocol using a single DNA oligonucleotide with NEBuilder HiFi DNA Assembly Master mix is a simple and streamlined way to rapidly create specifically targeted Cas9/sgRNA plasmids.
Help us celebrate our 50th anniversary! We have hidden 1,000 golden butterflies and are waiting for you to find them. They can be anywhere that you find NEB! Beginning April 15th, be sure to visit our website and tables at tradeshows and events you are attending. Visit our social media channels frequently for tips on where we have hidden the butterflies – and once you find one, either click or scan the code to be eligible for a 50th anniversary prize pack, as well as a grand prize trip to NEB headquarters in Ipswich, MA!.
To save your cart and view previous orders, sign in to your NEB account. Adding products to your cart without being signed in will result in a loss of your cart when you do sign in or leave the site.