The pGPU6/GFP/Neo expression vector was purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The enzymes (T4 DNA ligase, BamH I, Bbs I, and Pst I enzymes), Escherichia coli JM109 cells, and Agarose Gel DNA Purification Kit Version 2.0 were purchased form Takara Biomedical Technology Co., Ltd. (Beijing, China). The plasmid kit was purchased form Sigma Co., LLC. (Shanghai, China). The Lipofectamine 2000 and TRIzol reagent were purchased from Invitrogen Applied Biosystems (Shanghai, China). The primary antibodies (HIF-2α and P-gp) and the goat anti-mouse HRP-conjugated secondary antibody were purchased from Abcam (Shanghai, China). MTT was purchased form Dojindo Laboratories (Kumamoto, Japan).
Bbs 1
Manufactured by Takara Bio
Sourced in China
The Bbs I is a laboratory equipment product developed by Takara Bio. It serves as a core function for certain applications, though a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using bbs 1
1
Plasmid Construction and Cell Transfection
2
CRISPR Interference for Enhancer Repression
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CRISPR interference (CRISPRi) mediated enhancer repression was performed with pX330a Cas9-KRAB vector (Addgene #92361, www.addgene.org ) [63 (link)]. CRISPOR (http://crispor.tefor.net/ ) was used to design short guide RNAs (sgRNAs). Twenty microliters of primer pair (100 μmol/L) and 80 μL H2O were heated at 95 °C for 5 min and cooled down to RT to form dimer. The pX330a Cas9-KRAB vector was digested with Bbs I (TaKaRa, Dalian, Liaoning, China) and recovered with gel extraction. Next, the products were ligated with the dimer using T4 DNA ligase (TaKaRa, Dalian, Liaoning, China) at 16 °C o/n. The confirmed recombinant vectors were transfected into PBMs at 50% ~ 60% confluence which grew in 6-well plates and cells were collected after 24 h incubation.
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