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

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

2.2 Construction of plasmid miR130a-CRISPR
The oligonucleotides for site-specific chromatin cleavage of miR-130a target regions (Table 1) were
designed using a bioinformatics filtering tool (http://crispr.mit.edu/). Oligonucleotides used to target sitespecific chromatin cleavage were: miR130a-3p (3p) forward 5'-CACCGTGCAATGTTAAAA GGGCAT3', reverse 5'-AAACATGCCCTTTTAACATTGCAC-5'; miR130a-5p (5p) forward 5'-CAC
CGCACAATGTGAAAAGAGCTC-3', reverse 5'-AAACGAGCTCTTTTCACATTGTGC-3'; and
miR130a-stem loop (SL) forward 5'-CACCGTGTAACACGATGACAGACG-3', reverse 5'-
AAACCGTCTGTCATCGTGTTACAC-3'.
Each pair of oligonucleotide was phosphorylated and annealed at 37 °C (30 min) followed by 95 °C (5
min and then ramp down to 25 °C at 5 °C /min) in a thermal cycler (Bio-Rad MyCycler Thermal Cycler
PCR). The annealed oligonucleotides were diluted at 1:200 with RNase-free water and cloned into the BpiI
sites of pSpCas9(BB)-2A-GFP (Cp458) plasmid [13]. The plasmid was a gift from Feng Zhang (Addgene
plasmid # 48138). The miR130a-CRISPR plasmids were then transformed into stable competent E.coli
(NEB C040H) according to manufacturer’s protocol. The transformed cells were spread on LB agar with
ampicillin (100 µg/mL) and incubated overnight at 37 °C.

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Discussion

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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