Human embryonic kidney HEK293 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and penicillin, streptomycin, and all cell culture reagents were obtained from Carlo Erba Reagents (Cornaredo, Milano, Italy). Enzymes for PCR and the cloning procedure (FastDigest, FastDigest Green Buffer, FastAP Thermosensitive Alkaline Phosphatase, T4 DNA ligase and Phusion high-fidelity DNA polymerase) were obtained from ThermoFisher Scientific (Waltham, MA, USA). The reporter vectors pGL3-SV40-LgBiT-hAR and pGL3-SV40-hAR-SmBiT were obtained by standard molecular cloning procedures. The PureYieldTM Plasmid Miniprep System kit, gel, and PCR extraction kit were obtained from Promega (Promega, Madison, WI, USA). The Nano-Glo® Live Cell Assay System substrate and FuGENE® HD transfection reagent were obtained from Promega (Promega, Madison, WI, USA). The 96-well microspace round-bottom cell culture plates were obtained from Elplasia (Kuraray, Tokyo, Japan). Testosterone and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Fastdigest green buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
FastDigest Green Buffer is a ready-to-use solution designed for DNA digestion reactions. It contains a tracking dye and loading buffer, which facilitates the visualization and loading of samples onto agarose gels for electrophoresis.
Automatically generated - may contain errors
Lab products found in correlation
Fastap thermosensitive alkaline phosphatase, by Thermo Fisher Scientific (2 mentions)
Sexai, by Thermo Fisher Scientific (2 mentions)
Hincii, by Thermo Fisher Scientific (2 mentions)
Gel doc xr imaging system, by Bio-Rad (2 mentions)
Ecori, by Thermo Fisher Scientific (2 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Pureyield plasmid miniprep system kit, by Promega (1 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
Fastdigest, by Thermo Fisher Scientific (1 mentions)
Testosterone, by Merck Group (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Hek293 cell, by American Type Culture Collection (1 mentions)
Fastdigest restriction enzyme, by Thermo Fisher Scientific (1 mentions)
Nucleospin gel and pcr clean up, by Macherey-Nagel (1 mentions)
Hotstart it fidelitaq pcr master mix 2x, by Thermo Fisher Scientific (1 mentions)
Fastdigest bamhi, by Thermo Fisher Scientific (1 mentions)
Generuler 1 kb dna ladder, by Thermo Fisher Scientific (1 mentions)
Generuler 1 kb plus dna ladder, by Thermo Fisher Scientific (1 mentions)
Thermal block, by Eppendorf (1 mentions)
Acci restriction enzyme, by New England Biolabs (1 mentions)
11 protocols using fastdigest green buffer
1
Androgen Receptor Transcriptional Assay
2
Plasmid Digestion and Purification
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5 μg of plasmids was digested in a 50 μl reaction containing FastDigest Green Buffer (Thermo Scientific), 3 μl FastDigest restriction enzyme (Thermo Scientific) and 1 μl FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific) (1 U/μl) for 30 minutes at 37°C. After separation on 1% agarose gel, the desired fragments were isolated using NucleoSpin Gel and PCR Clean-up (Macherey-Nagel).
3
PCR Amplification and RFLP Analysis of CASR Gene Exon 7
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For a 25 µl PCR reaction, 2 µl genomic DNA (100 ng/µl), 12.5 µl HotStart-IT® FideliTaq™ PCR Master Mix (2X) (catalog no. 71156, Affymetrix, USA), 8.5 µl RNase free water, and 1 µl of (100 pmol/ul) of forward primer:5’-CTGAGCTTTGATGAGCCTCAGAAGGAC-3’and reverse primer: 5’-CACTGATGACAAGCTCTGTGAACTGGA-3’)were used. The tubes that contain the PCR mixture were centrifuged at 5000 rpm for 10 min. The PCR thermocycler conditions were as follows: initial denaturation at 94°C for 5 minutes. Followed by 35 cycles of denaturation at 94°C for one minute, annealing at 63°C for one minute, and finally an extension at 72°C for one minute. A final extension step was performed at 72°C for 5 minutes.13 (link)The samples were then run on 2% agarose gel with ethidium bromide to visualize the amplified PCR products (269 bp).The genotype distributions of exon 7 polymorphism (rs1801725) in CASR gene were determined by restriction fragments length polymorphism (RFLP) procedure. In Eppendorf tube, 10µl of PCR product, 17µl nuclease-free water, 2µl 10x FastDigest green buffer and 1µl of Thermo Scientific FastDigest Hin1I (catalog no. FD0474, Thermo Scientific, USA) were added. Then the mixture was mixed by pipetting gently, and was spin down for few seconds. The incubation was done at 37°C in a heat block for 20 minutes with no inactivation step.
4
Generating Force Clamps with BamHI
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In order to generate force clamps with 0 pN force, the spring strand was cleaved with a BamHI restriction endonuclease. To this end, 200 µM of the scaffold DNA and 3× molar excess of BamHI_comp strand were hybridised in FastDigest Green buffer (Thermo Scientific) by heating the sample to 90 °C, followed by gradual cooling to 20 °C over 2 h. Afterwards, 1 U of FastDigest BamHI (Thermo Scientific) was added, incubated at 37 °C for 4 h. Subsequently, BamHI was heat inactivated at 80 °C for 10 min.
5
Restriction Digestion Protocol
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Restriction digestions were carried out using the restriction enzymes EcoRI, HincII, NsiI, PstI, SalI, ScaI, SexAI, and SpeI (Thermo Fischer Scientific). These enzymes gave several well separated bands when in silico digested by the NEBcutter (http://nc2.neb.com/NEBcutter2/ ). The digestions were carried out in a final volume of 10 µL, containing DNA (ca. 300 ng), 0.5 µL of enzyme and 1 µL of Fast digest green buffer (10×, Thermo Fisher Scientific). After 2–16 h incubation at 37 °C the restriction fragments along with undigested DNA and GeneRuler 1 kb DNA Ladder (Thermo Fisher Scientific) were loaded on 1% (w/v) agarose gel including 0.005% (w/v) Midori green. After the electrophoresis the fragments were visualized using UV transillumination and images recorded using the BioRad GelDoc XR+ imaging system.
6
Restriction Enzyme Digestion Analysis
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Restriction digestions were carried out using the restriction enzymes EcoRI, HincII, NsiI, PstI, SalI, ScaI, SexAI, and SpeI (Thermo Fischer Scientific). These enzymes gave several well separated bands when in silico digested by the NEBcutter (http://nc2.neb.com/NEBcutter2/ (accessed on 8 May 2021)). The digestions were carried out in a final volume of 10 µL, containing DNA (ca. 300 ng), 0.5 µL of enzyme, and 1 µL of Fast digest green buffer (10×, Thermo Fisher Scientific). After 2–16 h incubation at 37 °C, the restriction fragments along with undigested DNA and GeneRuler 1 kb plus DNA Ladder (Thermo Fisher Scientific) were separated on 1% (w/v) agarose gel and detected with ethidium bromide staining using UV transillumination. Images were recorded using the BioRad GelDoc XR+ imaging system.
7
PCR Amplification and Sau96I Digestion
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The PCR composed as for the above-mentioned sequencing reaction, but using the E4F2-E4R primer pair to amplify a part of exon 4 and intron 4 (Table 1). A 5µL PCR product was digested with 1uL FastDigest Sau 96I (Cfr13I, ThermoFisher) in 1× FastDigest Green Buffer at 37°C for 12 minutes. The results of digestion were visualized by gel electrophoresis.
8
Genotyping of NUDT15 and TPMT Variants
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PCR-RFLP genotyping for the two loci was performed as follows. The target region of the NUDT15 and TPMT genes was amplified by PCR using the primers PCP-0023/24 and PCP-0027/28 with the stepdown PCR protocol as described above. Each NUDT15 c.415C>T genotyping restriction digestion reaction contained 1.7 µL unpurified PCR product, 0.2 µL FastDigest Taal restriction enzyme (Thermo Fisher, Waltham, MA, USA), 0.3 µL 10× FastDigest Green Buffer (Thermo Fisher, Waltham, MA, USA) and nuclease-free water added up to 5 µL. Each TPMT*3C genotyping restriction digestion reaction contained 1.7 µL unpurified PCR product, 0.2 µL AccI restriction enzyme (New England Biolabs, Ipswich, MA, USA), 0.3 µL CutSmart Buffer (New England Biolabs, Ipswich, MA, USA) and nuclease-free water added up to 5 µL. The digestion mix was incubated either at 65 °C (for TaaI digestion) or 37 °C (for AccI digestion) for 15 min on a thermal block (Eppendorf, Hamburg, Germany), and separated alongside 2.0 µL of undigested PCR product using a 2% agarose gel in 1× TBE buffer by electrophoresis at 100 V for 45 min.
9
Synthesis and Purification of EGFP mRNA
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In vitro transcription (IVT) EGFP plasmid was made by associate professor Rasmus O. Bak, Department of Biomedicine, Aarhus University. EGFP mRNA was generated by IVT. First, the EGFP plasmid was linearized by mixing 15 µL nuclease-free water (Thermo Scientific, Waltham, MA, USA, #R0582), 2 µL 10× Fast Digest Green Buffer (Thermo Scientific, #00959802), 2 µL (1 µg/µL) EGFP plasmid, and 2 µL Fast Digest restriction enzyme BbsI (Thermo Scientific, #00986235) and stored on ice. The reaction mixture was incubated at 37 °C for 3 h. Next, the linearization of the EGFP plasmid was visualized on a 1% agarose gel with a 1 kb marker (Thermo Scientific, #SM0311). NucleoSpin Gel and PCR Clean-up (Macherey, #2006/001) was used to elute the EGFP DNA from the agarose gel by following the manufacturer’s protocol. Finally, IVT was performed by using MEGAscript kit (Thermo Fischer Scientific, #AMB13345) following manufacturer’s protocol with one improvement as 3 µL CleanCap AG (6 mM) (TriLink Biotechnologies, San Diego, CA, USA, #N-7113-5) was added directly to the reaction mix to increase the stability of the mRNA and its translation. The RNA concentration was measured by a Nanodrop 1000 Spectrophotometer. The EGFP mRNA was stored at −20 °C.
10
Plasmid Linearization by Restriction Digest
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