For most experiments, cells were cultured in YEA medium that contains phosphate. In experiments comparing levels of pho1 expression in the presence or absence of phosphate, EMM was used with or without 15.5 mM sodium phosphate and 20 mM potassium phosphate. Total RNA was isolated by incubating cells in hot phenol heated to 65 °C for 10 min followed by 3 additional extractions using phenol-chloroform. RNA was precipitated using the sodium-acetate-ethanol method. Northern blots were performed according to the published protocol6 (link). 10 µg of RNA was resolved on a 1% formaldehyde-agarose denaturing gel and capillary transferred using NorthernMAX transfer buffer (Thermo Fisher Scientific) onto positively charged BrightStar-Plus nylon membrane (Ambion) and crosslinked using UV Stratalinker 2400 (Stratagene). The T7 in vitro transcription kit (Promega) was used to generate α-P32-UTP (PerkinElmer) labeled RNA probes (Supplementary Table 2 ) that were hybridized to the membrane overnight at 65 °C in ULTRAhyb buffer (Ambion). The membrane was exposed and scanned using a Typhoon FLA 9500 phosphor imager (GE Healthcare).
T7 in vitro transcription kit
Manufactured by Promega
Sourced in United States
The T7 in vitro transcription kit is a laboratory tool used for the production of RNA from DNA templates. It utilizes the T7 RNA polymerase enzyme to transcribe DNA sequences into RNA molecules. The kit provides the necessary components, including the T7 polymerase, nucleotides, and reaction buffers, to facilitate this in vitro transcription process.
Automatically generated - may contain errors
Lab products found in correlation
Typhoon fla 9500 phosphorimager, by GE Healthcare (2 mentions)
Brightstar plus nylon membrane, by Thermo Fisher Scientific (2 mentions)
Ultrahyb buffer, by Thermo Fisher Scientific (2 mentions)
Northernmax transfer buffer, by Thermo Fisher Scientific (2 mentions)
Immunoprecipitation lysis buffer, by Beyotime (1 mentions)
Protein loading buffer, by Sangon (1 mentions)
Coomassie brilliant blue, by Beyotime (1 mentions)
Mirvana mirna isolation kit, by Thermo Fisher Scientific (1 mentions)
Dynabead, by Thermo Fisher Scientific (1 mentions)
All in onetm mirna qrt pcr detection kit, by GeneCopoeia (1 mentions)
Fugene hd transfection reagent, by Promega (1 mentions)
Qubit 3, by Thermo Fisher Scientific (1 mentions)
Pgem t, by Promega (1 mentions)
Rneasy column, by Qiagen (1 mentions)
Cm1950, by Leica (1 mentions)
Enhanced sensitive ish detection kit, by Boster Bio (1 mentions)
11 protocols using t7 in vitro transcription kit
1
RNA Extraction and Northern Blot Analysis
2
Identification of LHFPL3-AS1 RNA Interactome
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The DNA sequence of LHFPL3-AS1-long or LHFPL3-AS1-short was amplified with sequence-specific primers containing T7 RNA polymerase promoter sequence. Then the purified PCR product was used as the template for in vitro transcription. The RNA transcript was synthesized using T7 in vitro transcription kit (Promega, USA) and biotinylated with EZ-Link Biotin kit (Thermo scientific, USA) according to the manufacturer’s instructions. The biotin-labeled RNAs were purified with mirVana miRNA Isolation Kit (Ambion, USA).
Cancer stem cells (5 × 106) were lysed using immunoprecipitation lysis buffer (Beyotime, China) containing 2 mM protease inhibitor. After centrifugation at 300×g for 5 min, the cell lysate was incubated with the biotinylated sense or antisense LHFPL3-AS1 RNA at 4 °C overnight. Subsequently, the mixture was incubated with streptavidin-conjugated Dynabeads (Thermo Scientific, USA) on a rotator for 2 h at 4 °C. The beads were washed with lysis buffer and then boiled in protein loading buffer (Sangon Biotech, Shanghai, China). The proteins were separated by gel electrophoresis and stained with Coomassie brilliant blue (Beyotime, China). The separated proteins bands specific for the sense LHFPL3-AS1 RNA were excised for mass spectrometry analysis.
Cancer stem cells (5 × 106) were lysed using immunoprecipitation lysis buffer (Beyotime, China) containing 2 mM protease inhibitor. After centrifugation at 300×g for 5 min, the cell lysate was incubated with the biotinylated sense or antisense LHFPL3-AS1 RNA at 4 °C overnight. Subsequently, the mixture was incubated with streptavidin-conjugated Dynabeads (Thermo Scientific, USA) on a rotator for 2 h at 4 °C. The beads were washed with lysis buffer and then boiled in protein loading buffer (Sangon Biotech, Shanghai, China). The proteins were separated by gel electrophoresis and stained with Coomassie brilliant blue (Beyotime, China). The separated proteins bands specific for the sense LHFPL3-AS1 RNA were excised for mass spectrometry analysis.
3
SARS-CoV-2 S and HA Gene Expression
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S and HA gene fragments, 1089 bp and 1111 bp respectively, were cloned into pcDNA3.1+ expression vector. The merged plasmid containing both S-gene and HA-gene was cloned, with S-gene is on the 5′ end, and the HA on the 3′ side. RNA transcription was conducted using T7 invitro transcription kit (Promega). The Gene-S and HA sequences were obtained from the 2019 nCoV (SARS CoV2) whole genome sequence (GenBank: MN908947 and GenBank: CY249803.1 respectively).
4
In Vitro Synthesis and Kinetic Analysis of Ribozymes
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The DNA template of substrate ap11, which contains the 37 nucleotide long AP mRNA sequence, was amplified by PCR using pGEM3zf (+) as a template with forward primer AF25 (5′-GGAATTCTAATACGACTCACTATAG-3′) and reverse primer AP11 (5′-CGGGATCCGTCCGAGGACGACGACGACGCCGCCGCCCTATAGTGAGTCGTATTA-3′) which contains a T7 promoter and the AP coding sequence. Plasmids pFL117, pV718, pV718-C and pC102, which were described in previous studies [19 (link),27 (link)], were used as templates to generate ribozymes M1-A, V718-A, V718-C and M1-C, respectively. The forward primer was AF25 while the reverse primer was M1AP11 (5′-CCCGCTCGAGAAAAAATGGTGTCGTCGTCGTCCTCGGA TGTGGAATTGTG-3′) with the positions corresponding to the guide sequence underlined. Ribozymes M1-C and V718-C contained the same mutations found in C102 which is a non-functional M1 RNA mutant with point mutations (A347C348 → C347U348, C353C354C355G356 → G353G354A355U356). A T7 in vitro transcription kit (Promega) was used for synthesizing RNA substrate ap11 and ribozyme RNAs [28 (link)]. Kinetic analyses and gel-shift binding assays were carried out following experimental procedures as described [19 (link),20 (link),29 (link)].
5
NLRP4E Knockdown in Oocytes
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The NLRP4E template sequences for the siRNA are listed in Additional file 1 : Table S2. The siRNA was constructed, annealed, and purified using Promega’s T7 in vitro transcription kit (Cat #: P1700, USA). The purified siRNAs were kept at −80 °C. The final small interfering RNA (siRNA) was a mixture of siRNAs corresponding to four different regions at the same final concentration of 5 μM.
For NLRP4E knockdown in oocytes by siRNA, we used Millipore’s siRNA transfection system (Cat #: N2913, USA) following the method described in our previous report. During siRNA processing (usually 36–44 h), 2.5 μM milrinone was added to inhibit meiosis.
For NLRP4E knockdown in oocytes by siRNA, we used Millipore’s siRNA transfection system (Cat #: N2913, USA) following the method described in our previous report. During siRNA processing (usually 36–44 h), 2.5 μM milrinone was added to inhibit meiosis.
6
Nitrogen Starvation Induces sme2 Expression
7
Suppression of lncR26319 Expression Using ASO
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Antisense oligonucleotides (ASO) (TaKaRa, Dalian, China) were synthesized to induce the suppression of lncR26319 expression. The lncR26319 ASO was designed as a gapmer uggugTTGTTAAGGGgcuuc, where the 1st and last 5 nucleotides were ribonucleotides with 2′‐O‐methyl modification flanking 10 deoxyribonucleotides. The total probe contained a phosphorothioate modification. The lncR26319 ASO was transfected into the BmN4 cells with liposome (FuGENE® HD Transfection Reagent, Promega, Madison, WI, USA). The double‐stranded RNA (dsRNA) of EndoA and EGFP was designed using the online software of SnapDragon (https://www.flyrnai.org/cgi‐bin/RNAi_find_primers.pl ) and synthesized with T7 in vitro transcription kit (Promega, Madison, WI, USA). The dsRNA of EGFP was used as a negative control for dsRNA experiments. An amount of 2 μg dsRNA or 50 μmol/L ASO was transfected into the BmN4 (70% confluence) cells in 12‐well plates by liposome transfection method. Silencing efficiency of lncR26319 or EndoA was determined after 48 h of transfection by qRT‐PCR; actinA3 was used as an internal control. The All‐in‐OneTM miRNA qRT‐PCR Detection Kit (GeneCopoeia, Rockville, MD, USA) was used to detect the expression changes of miR‐2834. U6 was used as an internal control.
8
Generating LASV Positive Controls
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LASV strains (Pinneo, ISTH1121-NIG-2012, Nig08-A19, Josiah, Ouoma-R123 and Togo/2016/7082) belonging to lineages I to VI, respectively, were randomly selected as six positive templates (5 (link)). The detail information of these positive templates, including strain name, GenBank accession number, and amplification region is listed in Table 2 . The DNA sequences in target region were synthesized by Sangon Biotech (Shanghai, China) Co., Ltd., and then cloned into T7 polymerase expression vector pGEM-T (Invitrogen, USA). The pGEM-T plasmids were used as a template to be in vitro transcribed into RNA according to T7 in vitro transcription kit (Promega, USA). The RNA was purified with RNeasy columns (Qiagen, Germany) and quantified using Qubit 3 (Invitrogen, USA) as final positive controls.
9
Localization of HcCPOX Expression in Purple Mussel Mantle
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In situ hybridization was used to detect the precise locations of expression of HcCPOX in the mantle of purple mussels. First, the RNA probes were synthesized using the T7 in vitro Transcription Kit (Promega, USA), according to the manufacturer’s instructions. The mantle samples (including outer fold, middle fold, inner fold of the mantle) were then collected and fixed in 4% paraformaldehyde for 6 h at 4°C, and then placed in 20% sucrose overnight at 4°C [5 (link), 7 (link)]. A freezing microtome (Leica CM 1950; Wetzlar, Germany) was used to slice mantle samples into 13-μm thicknesses and stored at −80°C. Finally, in situ hybridization was performed according to the manufacturer’s instructions (Enhanced Sensitive ISH Detection Kit, Boster, Switzerland).
10
Virus-Induced Gene Silencing to Study Stripe Rust in Wheat
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For Barley Stripe Mosaic Virus (BSMV) assay in wheat, two silencing fragments (VIGS-1, VIGS-2) were designed and combined to specifically knock down TaBIR1 transcript. Each fragment was evaluated by BLASTn against wheat genome to ensure specific silencing. Both fragments were cloned and inserted into BSMV:γ vector as previously described [53 (link)]. Plasmids were linearized and transcribed using the T7 in vitro transcription kit (Promega) following the manufacturer’s instructions. The transcripts of BSMV:TaBIR1 (containing VIGS-1, VIGS-2) mixed with BSMV:α, BSMV:β, and FES buffer were inoculated on the second leaves of two-leaf stage wheat seedlings. TaPDS encoding T. aestivum phytoene desaturase was used as silencing index. A fragment of GFP was used as negative control. After virus inoculation, infected plants were maintained in a controlled growth chamber at 25 °C. Twelve days later, the fully expanded fourth leaves were further inoculated with fresh Pst CYR23 urediospores and maintained at 16 °C. The Pst inoculated leaves were collected at 0, 24, 48 hpi for silencing efficiency and histochemical assays. Samples at 240 h for fungal biomass was analyzed as described [54 (link)].
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