hCas9

CRISPR Mouse - Deletion C2C12 Dmd

Experiment
CRISPR Mouse - Deletion C2C12 Dmd
Product
hCas9 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.
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 EGFP-2A-cas9, i20-gRNA, and i23-gRNA adenoviruses. EGFP-2A-cas9, i20-gRNA, and i23-gRNA cassettes were subcloned into pShuttle-CMV vector (Clontech, Mountain View, CA) and recombinant adenovirus genomic DNA were generated using the AdEasy-1 Adenovirus system (Agilent Technologies, La Jolla, CA) according to the manufacturer's instructions. The adenoviral particles were packaged and amplified in AD293 cells and purified by cesium chloride gradient ultracentrifugation followed by dialysis in storage buffer (10 mmol/l Tris-HCl pH 8.0, 2 mmol/l MgCl2, 4% sucrose). The titers of the adenovirus preparations were quantified by measuring the OD260.29

Cell culture and transfection. C2C12 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and electroporated with Neon Transfection System (Invitrogen, Carlsbad, CA). Briefly, 1 × 105 cells were electroporated with 0.25 µg cas9 and 0.125 µg i20-gRNA and 0.125 µg i23-gRNA plasmids. The electroporation conditions were 1,650 V, 10 ms, 3 pulses. Primary mdx myoblasts were isolated from the hind-limb skeletal muscles of mdx mice of 6 weeks old by digestion with collagenase type IA (Sigma-Aldrich, St Louis, MO), and cultured in DMEM/F-12 supplemented with 20% FBS. mdx myoblasts at 50–60% confluence were infected with EGFP-2A-cas9, i20-gRNA, and i23-gRNA adenoviruses at 100 multiplicity of infection. After 48 hours, C2C12 and mdx myoblasts were collected for the following genomic DNA analysis. Electroporated C2C12 cells were cultured in differentiation medium. mdx myotubes were harvested to analyze the dystrophin expression by RT-PCR after 3 days in differentiation medium.

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

Reviews

hCas9 from Addgene has not yet been reviewed for this experiment

We'd love it if you would be the first to write a review!

Discussion

Discussion

1 year 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

2 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 C2C12 Dmd using hCas9 from Addgene.

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

Manufacturer protocol

Download the product protocol from Addgene for hCas9 below.

Download PDF Download manufacturer protocol

Videos

Check out videos that might be relevant for performing CRISPR Mouse - Deletion C2C12 Dmd using hCas9 from Addgene. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.

We haven't found any additional videos for this experiment / product combination yet.

Become shareholder Discussions About us Contact Privacy Terms