Plasmids used in this manuscript are listed in table S1. LCB constructs were generated by Gibson assembly (New England Biolabs) of fragments LexA [from pDN92 (17 (link))], CIB1 [from LITE2.0 TALE-CIB1 (19 (link))], and biLINuS [from pDN92 (17 (link))] following the manufacturer’ s instructions. CRY2PHR-VPR constructs were generated by Gibson assembly of fragments CRY2PHR [from LITE2.0 CRY2PHR-VP64 (19 (link), 48 (link))] and VPR [from SP-dCas9-VPR (27 (link))]. DNA fragments were amplified by polymerase chain reaction (PCR) using Q5 DNA polymerase (New England Biolabs). DNA oligos for PCR amplification were synthesized by Integrated DNA Technologies. PCR products were purified by the agarose gel electrophoresis method (D4001; Zymo Research). Purified DNA fragments were cloned into pCAGGS vector (for transient transfection) or pHR-PGK (for transient transfection or lentiviral infection). Light-inducible reporter constructs were generated by cloning the target gene into the pDN100 vector (with the original reporter gene removed, for transient transfection) or pHR vector (with LexA binding site and minimal promoter, for lentiviral infection) using the Gibson assembly method. Constructs were confirmed by Sanger sequencing (Genewiz).
D4001
Manufactured by Zymo Research
The D4001 is a laboratory instrument designed for automated nucleic acid extraction. It utilizes magnetic bead-based technology to efficiently purify DNA, RNA, or other nucleic acids from a variety of sample types, including cells, tissues, and bodily fluids. The instrument features a compact and user-friendly design, allowing for high-throughput processing and consistent results.
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Lab products found in correlation
3 protocols using d4001
1
Plasmid Construction for Light-Inducible Systems
2
RNA Seq Library Preparation and Sequencing
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Total RNA was extracted from each sample and purified using the Norgen Total RNA isolation kit and quantified using a NanoDrop Spectrophotometer ND-100 and quality control was assessed with bioanalyzer total RNA Nano kit. One μg of mRNA was fragmented to an average length of 200 bp by incubation for 5 min at 94 oC with 5X fragmentation buffer (Illumina, RS-100-0801). Efficiency of the fragmentation was defined on Bioanalyzer RNA Pico Chip. The fragmented mRNA was randomly primed and reversed transcribed using Super Script II cDNA synthesis kit (Invitrogen, 18064-014). After second-strand synthesis, the cDNA underwent end-repair and ligation reactions according to the Illumina mRNA-Seq Sample Prep Kit protocol. The cDNA library was size-fractioned on a 2% TBE agarose gel. Material in the 350–400 bp range was excised and purified (Zymo Research, D4001). Half of the eluted cDNA library was used as a template for amplification according to mRNA-Seq Sample Prep Kit protocol. The PCR product was purified using PureLink PCR micro purification kit (Invitrogen, Q32850). The library was then used to build clusters on the Illumina flow cell and analysis was done using Illumina Hiseq 2000 platform (Illumina, San Diego, CA, USA) at McMaster University to a target depth of 6 M reads per sample.
3
Construction of Nuclear-Targeted Fyn FRET Biosensor
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