SLC20A2 Human Gene Knockout Kit

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

Knockdown of SLC20A2 with CRISPR-Cas9
A CRISPR knock-down kit against human SLC20A2 was purchased from OriGene (USA, Cat# KN206029). Transfections were performed as recommended by the manufacturer with some alterations. Briefly, 2 × 105 SaOs-2 cells were seeded into 6 well plates and maintained for 24 hours. Lipofectamine 3000 (Invitrogen, Cat# L30000) was used at a final concentration of 0.5% together with a total of 5 μg plasmid (2.5 μg gRNA or Scrambled control with 2.5 μg donor) per well. Lipofectamine-DNA complexes were made up in Opti-MEM (Invitrogen, Cat# 31985070). Prior to transfection, medium was replaced with growing medium without penicillin-streptomycin. Cells were maintained for 24–48 hours before cells were returned to growth medium. Transfected cells were sub-cultured 5–7 times before puromycin selection (1 μg/ml, Invitrogen, Cat# A1113803) and confirmation of gene knockdown. Genomic PCR primers were designed against regions of the SLC20A2 gene flanking the guide-RNA target sequence and were: Forward 5′-CGCTGACTGAACACAACCAA-3′ and reverse 5′-CTAACTTCCCCAGCCATGAG-3′ to generate an amplicon of 487 bp in a reaction with 100 ng of genomic DNA extracted from cells with the DNeasy Blood and Tissue kit (Qiagen, USA, Cat# 69506). PCR products were run in an agarose gel for visualization or purification. For T7 digests, PCR bands were excised from agarose using the QIAquick Gel Extraction kit (Qiagen, Cat# 28704). A hybridization reaction was then performed in a PCR cycler with 200 ng of purified PCR product with 2 μl of NEB buffer 2 in a 20 μl reaction. Cycling conditions were 95 °C, 5 min; ramp down to 85 °C (−2 C/s); ramp down to 25 °C (−0.1 C/s); hold at 4 °C. T7 endonuclease (1 U, NEB, CAT# M0302) was added reaction−1 and incubated at 37 °C for 15 min. EDTA (2 μl of 0.25 M stock) was added to stop the reaction before samples were electrophoretically separated in an agarose gel.

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Manufacturer protocol

Download the product protocol from OriGene for SLC20A2 Human Gene Knockout Kit below.

Download manufacturer protocol

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