The NCoR1 and NCoR2 exon 37 constructs in pTBNde1 were grown in media and isolated (plasmid midi-prep kit, Machery-Nagel). Approximately 200 pg of plasmid at 20 pg nl−1 was injected into each X. laevis embryo at the two-cell stage, and the embryos grown to the mid neurula stage (Nieuwkoop and Faber, stage 16) [73 ]. Zebrafish embryos were injected at the one-cell stage with 200 pg of plasmid at 400 pg nl−1, grown overnight at 32°C and collected at stage 27/28 hpf. Total nucleic acid was then extracted [70 (link)] and DNA removed by RNAse-free DNAse digestion. The remaining RNA was precipitated, resuspended and converted to cDNA using reverse transcriptase. The splicing status of the transcripts from the clones was assayed by PCR using forward and reverse primers against the human exons [72 ] or one human and one Xenopus- or zebrafish-specific sequence and the products resolved on 1.5–2% agarose gels.
Plasmid midi prep kit
Manufactured by Macherey-Nagel
Sourced in Germany
The Plasmid midi-prep kit is a laboratory equipment designed for the purification of plasmid DNA from bacterial cultures. It is used to extract and isolate plasmid DNA in a medium-scale format.
Automatically generated - may contain errors
Lab products found in correlation
Pcdna3, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 goat anti rabbit igg h l, by Thermo Fisher Scientific (1 mentions)
Linear pei, by Polysciences (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Lb broth, by Thermo Fisher Scientific (1 mentions)
Toto 3, by Thermo Fisher Scientific (1 mentions)
Hyaluronic acid, by Lifecore Biomedical (1 mentions)
Rabbit anti active caspase 3 antibody, by BD (1 mentions)
Pgl3 basic, by Promega (1 mentions)
Ampicillin, by MDBio (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by MDBio (1 mentions)
2 protocols using plasmid midi prep kit
1
Analyzing Splicing of NCoR Transcripts
2
Versatile Nanocarrier Synthesis and Optimization
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Branch polyethylenimine (25kDa) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Linear PEI (20kDa) was purchased from PolyScience (Warrington, PA, USA). Paraformaldehyde (PFA) were purchased from Alfa Aesar (Ward Hill, MA, USA). β-cyclodextrin (β-CD), amiloride and chlorpromazine (CPZ) were purchased from TCI (Tokyo, Japan). The gene expression vectors were prepared by cloning TRAIL gene (Costumed synthesis by Genedirex), mOrange or firefly luciferase reporter gene (pGL3-basic, Promega) into the downstream of CMV promoter of pcDNA3 (Invitrogen) vector. Each pDNA was amplified in Escherichia coli (DH5-α) and purified using plasmid midiprep kit (Macherey Nagel, Germany). TOTO-3, LB broth and Alexa fluor ® 594 goat anti-rabbit IgG (H+L) were purchased from Life Technologies (Gaithersburg, MD, USA). Hyaluronic acid (HA) (100 kDa) was purchased from Lifecore Biomedical, Rabbit anti-active caspase-3 antibody and Transwell were purchased from BD. MTT and ampicillin were purchased from MDBio.
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