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

Protocol tips

Upstream tips	Protocol tips	Downstream 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.	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.

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.

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Papers

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Manufacturer protocol

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

Download manufacturer protocol

Videos

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