The above oligonucleotides were annealed and added to a transcription reaction so that the final concentration was 0.1 μg/mL. Final concentrations of other components were 1× transcription buffer (Thermo Fisher), 10 mM DTT, 0.3 mM each ATP, GTP, and UTP, 0.5 pmol/ml alpha-32P CTP (3000Ci/mmol), 0.02 mM CTP, T7 RNA polymerase (Thermo Fisher). After 2 h incubation at 37°C, additional T7 polymerase was added and incubation continued for another 2 h. RQ1 DNase buffer was then added to 1× final concentration along with RQ1 DNase (Promega) and incubated at 37°C for 20 min. The RNA was ethanol precipitated and resuspended in TE. This was mixed 1:1 with formamide loading dye (93% formamide, 30 mM EDTA, 0.1× TBE, 0.5% bromophenol blue, 0.5% xylene cyanol), heated to 95°C for 5 min and loaded on a 8% acrylamide 7 M urea gel buffered with 1× TBE. After electrophoresis, bands were cut out of the gel and the RNA was extracted with 0.3 M sodium acetate at room temperature for 1 h. Samples were centrifuged briefly, liquid was removed, transferred to a Spin-X column (Corning), centrifuged, and ethanol precipitated. This procedure was then repeated. After ethanol precipitation, the pellet was resuspended in H2O.
Rq1 dnase buffer
Manufactured by Promega
Sourced in United Kingdom
RQ1 DNase buffer is a solution designed to facilitate the removal of DNA from RNA samples. It provides the necessary components to activate the RQ1 RNase-Free DNase enzyme, which is commonly used in RNA purification protocols to eliminate any contaminating DNA.
Automatically generated - may contain errors
Lab products found in correlation
Rq1 dnase, by Promega (4 mentions)
Transcription buffer, by Thermo Fisher Scientific (1 mentions)
T7 polymerase, by Promega (1 mentions)
Spin x column, by Corning (1 mentions)
T7 rna polymerase, by Thermo Fisher Scientific (1 mentions)
Sv gel and pcr clean up system, by Promega (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Rnase a, by Qiagen (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Protoscript 2, by New England Biolabs (1 mentions)
Rq1 dnase enzyme, by Promega (1 mentions)
Blood and tissue dna extraction kit, by Qiagen (1 mentions)
Nebnext ultra 2 dna library prep kit, by New England Biolabs (1 mentions)
Qiamp viral rna mini kit, by Qiagen (1 mentions)
Proteinase k, by Qiagen (1 mentions)
5 protocols using rq1 dnase buffer
1
In Vitro Transcription of Radiolabeled RNA
2
Viral particle purification and sequencing
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Partial purification of potential viral particles from D.alata acc. 313 leaf samples was performed as described previously (Jones et al. 2001 ). Pellets were resuspended in 150 µl of 1X RQ1 DNase buffer (Promega), which was then treated with 15 U of RQ1 DNase (Promega) and 10.5 U of RNase A (Qiagen) at 37°C for 2 h to digest non-particle-protected nucleic acids. RNA was extracted with an RNeasy Plant Mini Kit (Qiagen). Random RT-polymerase chain reaction (PCR) amplification was then performed with the TransPlex® Whole Transcriptome Amplification (Sigma-Aldrich) kit according to the manufacturer’s protocol. Potential RNA and DNA virus genome amplicons ranging in size from 200 to 1,000 bp were gel purified (SV Gel and PCR Clean-Up System (Promega)) and inserted into pGEM®-T Easy vector as recommended by the manufacturer (Promega). The inserts were amplified by PCR using the universal primers T7 and SP6, and fragments >250 bp were sequenced by single-pass double-stranded analysis (Cogenics) using the same primers. Sequence similarity searches were performed using BlastN and BlastX methods (Altschul et al. 1990 (link)).
3
Transcriptional Repression of Rps3-TAP
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Transcriptional repression of dox-repressible Rps3-TAP was assessed by inoculating YPGal media [with or without dox (0.2 μg/ml)] with a preculture grown in YPD to mid-log phase. Ten milliliters of cells was harvested at different time points. Harvest cells were resuspended in 400 μl of TES [10 mM tris HCl (pH 7.5), 10 mM EDTA, and 0.5% (w/v) SDS] buffer, and total RNA was isolated by hot-phenol extraction. After RNA extraction, each sample was precipitated and resuspended in 50 μl of deoxyribonuclease (DNase) master mix [5 μl of 10× RQ1 DNase Buffer (Promega), 1 μl of RQ1 DNase enzyme (Promega), and 44 μl of dH2O] to further remove DNA. Then, each sample was resuspended in 100 μl of H2O, and total RNA concentration was measured. One microgram of total RNA was used for reverse transcription using Protoscript II (New England Biolabs) per the manufacturer’s instructions, generating complementary DNA. Quantitative polymerase chain reaction (qPCR) was performed with Excella 2× SYBR master mix per the manufacturer’s instructions using primers listed in table S3.
4
Chlamydia trachomatis L2/434 Genome Sequencing
5
RNA Extraction and DNase Treatment
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Samples (100 μL) were incubated with 10 μL of Proteinase K (Qiagen UK) for 15 minutes at room temperature, then incubated at 70°C for 15 minutes to inactivate enzyme. Ten microliters of RQ1 DNase buffer (Promega, UK) and 10 μL of RQ1 DNase (Promega, UK) were added, then incubated at 37°C for 30 minutes. RNA was extracted using the QIAmp Viral RNA Minikit (Qiagen, UK), and eluted in 60 μL of buffer AVE.
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