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

CRISPR Mouse - Deletion B16-F1 PC7

Experiment
CRISPR Mouse - Deletion B16-F1 PC7
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.
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 editing
The guide sequences 5’-TGCGACCACCCATAGCAACC-3’ or 5’-GCTGAGCTGGGCCGTACATC3’ targeting murine Furin or PC7 respectively near their start codon were cloned into the expression
vector PX458 containing GFP-tagged Cas9 (Ran et al., 2013). The resulting sgRNA/Cas9 expression
vector were transfected in B16F1 cells. After 24 h, the cells were trypsinized, washed with PBS and
resuspended in PBS/1% FBS for single cell sorting for GFP by FACS into 96-well plate containing
complete medium. Clonal cell lines were expanded and screened by anti-Furin by Western blot. Genomic
DNA from clones was purified using QuickExtract ™ DNA Extraction Solution (Epicentre), and the
region surrounding the protospacer adjacent motif (PAM) was amplified using PfuUltra II Fusion HS
DNA polymerase (Agilent) using the primers:
Furin forward: 5’-TTTTAGGCTCAGCCGTGAGG-3’,
Furin reverse: 5’-GTTACGGATCCCATCCCACC-3’,
PC7 forward: 5’- AGCAAGCACCTGGATGATG -3’,
PC7 reverse: 5’- CTCTTCAGCAGCGTTTGCTC-3’.
PCR products were purified using NucleoSpin® Gel and PCR Clean-up kits (Macherey-Nagel) and
cloned into pCR™4-TOPO® TA vector for sequencing. For each cell line, at least 12-15 bacterial colonies were expanded to sequence their plasmid DNA.

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

Reviews

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

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 B16-F1 PC7 using pSpCas9(BB)-2A-GFP (PX458) from Addgene.

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

Manufacturer protocol

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

Download PDF Download manufacturer protocol

Videos

Check out videos that might be relevant for performing CRISPR Mouse - Deletion B16-F1 PC7 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.

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

Outsource your experiment

Fill out your contact details and receive price quotes in your Inbox

  Outsource experiment
Become shareholder Discussions About us Contact Privacy Terms