The cdrS CRISPRi strain (expressing crRNA#2) was complemented with either the single gene HVO_0581 (ftsZ2) or with the complete operon HVO_0582-HVO_0581. Transformed strains were grown in minimal medium supplemented with tryptophan (0.25 mM) and harvested in exponential phase (OD650 ≈ 0.4 to 0.52). RNA was isolated as described before (64 (link)), separated on a 1% agarose gel, and transferred onto a nylon membrane (Pall membrane). For hybridization experiments, radioactively labeled PCR probes against the selected targets were generated using the DECAprime II DNA labeling kit and [α-32P]dCTP (Life Technologies, Thermo Fisher Scientific). Membranes were subsequently incubated with the labeled PCR fragments against the mRNA of the cluster HVO_B0192-HVO_B0193 and against the mRNA of HVO_0739.
α 32p dctp
Manufactured by Thermo Fisher Scientific
[α-32P]dCTP is a radiolabeled nucleotide analog used in various molecular biology applications. It contains the radioactive isotope 32P incorporated into the dCTP molecule, which can be used to label DNA or RNA for detection and analysis purposes.
Automatically generated - may contain errors
Lab products found in correlation
Decaprime 2 dna labeling kit, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Typhoon biomolecular imager, by Cytiva (1 mentions)
Klenow fragment exo, by Thermo Fisher Scientific (1 mentions)
Tnt t7 quick coupled transcription translation system, by Promega (1 mentions)
Cyclone plus storage phosphor system, by PerkinElmer (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
6 protocols using α 32p dctp
1
Complementation of CRISPR Interference in H. volcanii
2
Quantitative analysis of RNA repression
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RNA was isolated from strains HV30 × pTA232-tele-0582anti#1, -#2, and -#3 and wild-type strain HV30 × pTA232 as described before (64 (link)). Ten micrograms of RNA was separated on a denaturing 8% polyacrylamide gel or a denaturing 1% agarose gel and transferred to a nylon membrane (Hybond-N+ membrane or Pall membrane). For hybridization experiments, radioactively labeled PCR probes against the desired targets were generated using the DECAprime II DNA labeling kit and [α-32P]dCTP (Life Technologies, Thermo Fisher Scientific). Membranes were subsequently incubated with the labeled PCR fragments.
To quantify the repression efficiency, Northern blotting membranes were exposed to imaging plates and analyzed with the Amersham Typhoon biomolecular imager and ImageQuantTL software. Signals were compared with the signals for the 16S rRNA used as a loading control. The amount of RNA signals detected for the wild-type controls was set to 100%. Northern blot analyses were done in triplicates.
To quantify the repression efficiency, Northern blotting membranes were exposed to imaging plates and analyzed with the Amersham Typhoon biomolecular imager and ImageQuantTL software. Signals were compared with the signals for the 16S rRNA used as a loading control. The amount of RNA signals detected for the wild-type controls was set to 100%. Northern blot analyses were done in triplicates.
3
Quantifying MM Cell RNA Expression
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MM cells in culture were incubated with transcription inhibitor actinomycin D (5 µg/ml) and cells (3×106) were collected at different time points over 12 hours, washed twice with cold PBS, RNA isolated (TRIZOL®, Invitrogen Corp, Carlsbad, CA) and quantified on a Synergy-H1 reader (BioTek Laboratories Inc, Winooski, VT), and 10–15 µg of RNA was used for northern blotting and hybridization as described previously30 . RT-PCR using forward primer 5’-TCGAAGTCGCACTGCTATGG – 3’ and reverse 5’-TCGAGAGATCCACTCTGGGG – 3’ on MM1.S total RNA was used to obtain a 389 bp amplicon that was labeled using α32P –dCTP (random primer DNA labeling system, Life Technologies, Grand Island, NY) for use as probe in northern blots.
4
Quantifying MM Cell RNA Expression
5
Protein-DNA Interaction Assay with CIC Fragments
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EMSA experiments were performed using CIC protein fragments synthesized with the TNT T7 Quick Coupled Transcription/Translation system (Promega). For expression of His-tagged proteins, bacterial cultures were induced for 2 h with 1 mM IPTG and proteins purified using the Proteus IMAC Mini Sample kit. DNA probes were synthesized as complementary oligonucleotides leaving 5’ GG overhangs, or amplified by PCR with primers carrying NotI restriction sites, subcloned, and released by NotI digestion. Probes were then end-labeled using α-32P-dCTP and Klenow Fragment, exo- (Thermo Scientific). The sequences of wild-type and mutant probes are shown in S1 Table .
Binding reactions were carried out in a total volume of 20 μl containing 60 mM Hepes pH 7.9, 20 mM Tris-HCl pH 7.9, 300 mM KCl, 5 mM EDTA, 5 mM DTT, 12% glycerol, 1 μg poly(dI-dC), 1 μg BSA, ~1 ng of DNA probe, and 1 μl of programmed or non-programmed (control) TNT lysate (or ~1 ng of bacterially expressed His-tagged protein). After incubation for 20 min on ice, protein-DNA complexes were separated on 5% non-denaturing polyacrylamide gels run in 0.5X TBE at 4°C, and detected by autoradiography.
Binding reactions were carried out in a total volume of 20 μl containing 60 mM Hepes pH 7.9, 20 mM Tris-HCl pH 7.9, 300 mM KCl, 5 mM EDTA, 5 mM DTT, 12% glycerol, 1 μg poly(dI-dC), 1 μg BSA, ~1 ng of DNA probe, and 1 μl of programmed or non-programmed (control) TNT lysate (or ~1 ng of bacterially expressed His-tagged protein). After incubation for 20 min on ice, protein-DNA complexes were separated on 5% non-denaturing polyacrylamide gels run in 0.5X TBE at 4°C, and detected by autoradiography.
6
Quantifying DNA Recombination Events
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DSBs, dHJs, crossovers, and non-crossovers were analyzed by DNA physical assays in combination with Southern blot as described previously (39–41 (link)). Briefly, the genomic DNA was extracted from synchronized yeast cells at desired time points in SPM and digested with Xho I. One-dimension (1D) gel electrophoresis was carried out, followed by a Southern blot to detect DSBs, crossovers, and non-crossovers. Two-dimension (2D) gel electrophoresis in combination with Southern blot was performed to detect IH-dHJs and IS-dHJs. For both crossovers and non-crossovers analysis, the genomic DNA was digested with Xho I and NgoM IV, separated by 1D gel electrophoresis, and detected by Southern blot. The probe for the Southern blot was labeled with α-32P-dCTP by a random labeling system (Thermo Fisher, Cat#18187-013). All DNA species in Southern blot were visualized using a phosphorimager (the Cyclone Plus Storage Phosphor System, PerkinElmer) and quantified using Quantity One software (Bio-rad).
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