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

CRISPR Mouse - Deletion ES (embryonic stem) cells MIR

Experiment
CRISPR Mouse - Deletion ES (embryonic stem) cells MIR
Product
pSpCas9(BB)-2A-GFP (PX458) from Addgene
Manufacturer
Addgene

Protocol tips

Upstream tips
To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip (20uL or 200uL one), puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate.

Publication protocol

CRISPR/Cas9 genome engineering
CRISPR/Cas9 genome engineering was performed as described (Ran et al. 2013). To delete the MIR genomic sequence, preserving its 3′ end region, we used two short guide RNAs (sgRNAs) (see Supplemental Fig. S4A). After cotransfection of mouse ES cells with pSpCas9(BB)-2A-GFP vectors containing sgRNA1 and sgRNA2 and selection by FACS, we screened for MIR deletions by PCR on genomic DNA of single-cell-derived ES cells with a pair of primers flanking the MIR (Supplemental Fig. S4B) and by sequencing of some of the PCR products (Supplemental Fig. S4C). We obtained different deletion lengths (Supplemental Fig. S4D).

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Reviews

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Discussion

Discussion

4 years ago

Author: Mario Udinese Italy

Floxing mice with CRISPR

Hi everyone! I am planning on floxing mice with CRISPR but I am having trouble deciding which region to target. Do you have any tips on choosing?

Discussion

5 years ago

Author: Ben Saar Israel

How to choose a region to target for CRISPR

Hi everyone! I am planning on floxing mice with CRISPR but I am having trouble deciding which region to target. Do you have any tips on choosing?

Share your thoughts or question with experts in your field by adding a discussion!

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing CRISPR Mouse - Deletion ES (embryonic stem) cells MIR using pSpCas9(BB)-2A-GFP (PX458) from Addgene.

Paper title
Transcriptional interference by RNA polymerase III affects expression of the gene
Full paper
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Manufacturer protocol

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

Download PDF Download manufacturer protocol

Videos

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