pSpCas9(BB)-2A-GFP (PX458)

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

CRISPR/Cas9 Plasmid Design and Construction
The GATA1 gene sequence was entered into the Zhang lab’s25 online generator (http://crispr.mit.edu/), and the appropriate CRISPR guide sequence that binds in close proximity to target (−2 bp upstream of Met1) was chosen. The CRISPR was constructed using standard cloning methods following the latest oligo annealing and backbone cloning protocol with single-step digestion-ligation. The CRISPR guide sequence was cloned into the pX458 backbone vector (plasmid 48138; Addgene), a human codon optimized pSpCas9 and chimeric guide RNA expression plasmid with a 2AEGFP, purchased through Addgene (https://www.addgene.org). Following construction, clones were verified by DNA sequencing by Eton Bio Incorporated.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing CRISPR Human - Deletion GATA1 using pSpCas9(BB)-2A-GFP (PX458) from Addgene.

Manufacturer protocol

Download the product protocol from Addgene for pSpCas9(BB)-2A-GFP (PX458) below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing CRISPR Human - Deletion GATA1 using pSpCas9(BB)-2A-GFP (PX458) from Addgene. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.