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12 protocols using prime a gene labeling kit

1

Southern Blot Analysis of Transgenic Rats

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Genomic DNA from the transgenic rats was digested with BamHI and EcoRV at 37 °C for 16 hours, and then separated on 0.8% agarose gels prior to Southern analysis. The probe was synthesized using the Prime-a-Gene Labeling kit (Promega, lot:0000182475), which used the Neo cassette as the template, and the probe was labeled with alpha-P32dATP.
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2

Northern Blot Analysis of pdm4 Mutant

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For northern-blot analysis, total RNA from wild-type and pdm4 seedlings was extracted and determined by using thermo NanoDrop 2000 (Thermo, USA). Three equal content RNA samples of the wild type and pdm4 mutant were separated on 1.3% (w/v) agarose-formaldehyde gels and subsequently blotted to a nylon membrane. Next, the membrane was hybridized with a specific probe labeled with 32P. The labeled probes were obtained by using the Prime-a-Gene Labeling Kit (SGMB01-Promega-U1100, USA). The sequences of the primers were according to Du et al. (2017) (link). All the analysis was performed at least in three independent repeats.
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3

Generating AAV Vectors for Pullulanase Expression

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To generate the pAAV-CB-Pull vector plasmid, we subcloned the KpnI-EcoRI fragment containing the HA-tagged Pullulanase cDNA from the pcDNA3.1-Pull vector into the pAAV-CB-hGAA vector41 (link) containing the CMV enhanced chicken β-actin hybrid (CB) promoter to replace the human GAA cDNA. To generate the pAAV-LSP-Pull vector, we cloned the ScaI-KpnI fragment containing the liver-specific promoter (LSP) from the pAAV-LSP-hGAA vector42 (link) into the pAAV-CB-Pull vector to replace the CB promoter. These vectors were packaged as AAV9 (AAV9-CB-Pull) and AAV8 (AAV8-LSP-Pull) in HEK293T cells using the calcium phosphate transfection method. The viral vectors were purified using iodixanol gradient ultracentrifugation method.43 (link) The titer of the viral stock was determined using purified viral DNA and Southern blotting with a biotin-labeled probe generated with Prime-A-Gene labeling kit (Promega, Madison, WI, USA). The viral vector stock was handled according to Biohazard Safety Level 2 guidelines published by the National Institutes of Health.
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4

Analysis of Ces5a gene expression

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Total RNA was extracted from tissue homogenates and cells with Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's recommendations. Northern blot analysis was performed according to a procedure described previously.18 (link) Twenty microgramme of total RNA from each sample were subjected to 1.0% (w/v) agarose-formaldehyde gel electrophoresis, blotted onto nylon membranes by capillary transfer and hybridized with a probe which was obtained from the full Ces5a clone digested with BamHI at both 291 and 1612 bp sites (Gene bank, NM_001012056). The32 (link) P-labeled probe was prepared by using the prime-a-gene labeling kit (Promega, Madison, USA). An 18S rRNA hybridization signal was used as a loading control.
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5

Quantitative analysis of mitochondrial mRNA

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Total RNA was isolated from 8-week-old flower buds using TRIzol reagent (Life Technologies) according to the manufacturer's instructions. RNA was treated with DNase Max (QIAGEN) when RNAs were used in reverse transcription (RT)-PCR or quantitative RT-PCR (qRT-PCR) assays. Mitochondrial mRNA abundances were measured by qRT-PCR. They were calculated using the comparative ΔΔCt method after normalization to the nuclear 18S ribosomal RNA as previously described in (15 (link)). Two biological and three technical repeats were performed for these analyses.
For RNA gel blotting, 10 μg of total RNA was electrophoretically separated in formaldehyde-containing (1.5% [w/v]) agarose gels and transferred onto nylon membranes (Genescreen) as described previously (10 (link)). Hybridization probes were generated by PCR amplification using gene-specific primers (listed in Supplementary Table S1) and radiolabeled using the Prime A Gene labeling kit (Promega) according to the manufacturer's recommendations.
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6

RNA Extraction and Analysis in Plant Mitochondria

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Total RNA was extracted from flower buds using the TRIzol reagent (Thermo Fisher Scientific), following the manufacturer’s instructions. To remove genomic DNA contamination from total RNA preparations, the DNase Max Kit (Qiagen) was used. For quantitative RT-PCR analysis, either 35 ng of total RNA was reverse-transcribed and amplified in a single tube with a volume of 15 µl using the QuantiTect SYBR Green RT-PCR Kit (Qiagen) or reverse transcription was performed separately on 1 μg of the total RNAs using a random hexamer. Quantitative RT-PCR for analyzing splicing efficiency in plant mitochondria was carried out, as previously described in Haili et al.54 (link), using the described set of primers. Two biological repeats and three technical repeats were performed on each genotype. cRT-PCR was performed as described in Forner et al.24 (link). RNA gel blot analysis was performed as previously indicated in Haili et al.54 (link). Shortly, 10 µg of total RNA was separated on a 1.5% (w/v) agarose gel. After electrophoresis, the RNAs were transferred to nylon membranes and cross-linked in a UV Stratalinker. Hybridization was performed with specific DNA probes amplified by polymerase chain reaction (PCR) and radiolabeled by the Prime A Gene labeling kit (Promega), following the manufacturer’s instructions.
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7

RNA Cleavage Fragment Analysis

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For cleavage fragment analysis, 20 μg of total RNA was loaded onto a 1% denaturing agarose gel and separated for 3–5 h at 120V. The RNA was blotted to an Amersham Hybond-NX nylon membrane (GE Healthcare Life Sciences) and UV-crosslinked (254 nm). After pre-hybridization in PerfectHybTM Plus Hybridization buffer (Sigma) for 1 h at 65ºC, the radioactively labeled probe (Prime-a-gene labeling kit, Promega) was hybridized overnight at 65ºC. Sequences for primers used for amplification of probe templates for GFP and endogenous miRNA targets are listed in Supplementary Table S4. After hybridization, the membrane was washed 3 times in 2xSSC (0.3 M NaCl, 30 mM sodium citrate), 0.1% SDS at 65ºC and developed by phosphorimaging. For Terminator (Epicentre) reactions, 10 μg of total RNA was incubated with 1 U Terminator (Epicentre) for 1 h at 30ºC. The reaction was terminated by phenol extraction and subsequent ethanol precipitation. The RNA was resuspended in 50% formamide and loaded with untreated RNA as control on a 1% denaturating agarose gel. Poly(A) RNA was purified from 10 μg of total RNA using Dynabeads Oligo(dT)25 (Life Technologies) according to the manufacturer's instructions.
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8

Production and Characterization of AAV-PHP.B Vector

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The AAV-PHP.B capsid plasmid was a gift from Dr. Benjamin Deverman of the California Institute of Technology.28 (link) The AAV-CB-hGAA vector74 (link) containing a universal CMV enhancer/CB) hybrid promoter, the hGAA cDNA, and the human growth hormone polyadenylation sequence was packaged as AAV-PHP.B in HEK293T cells using phosphate-mediated transfection; the viral vector was purified using the iodixanol gradient ultracentrifugation method.75 (link) The titer of the viral stock was determined using purified viral DNA and Southern blotting with a biotin-labeled probe generated with Prime-A-Gene labeling kit (Promega, Madison, WI, USA). The viral vector stock was handled according to Biohazard Safety Level 2 guidelines published by the NIH.
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9

RNA Extraction and Analysis for Plant Flower Buds

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Depending on the amount needed, total RNAs were isolated from flower buds using either TRIzol reagent (Life Technologies) or the RNeasy Plant Mini kit (Qiagen) following the manufacturer's recommendations and treated with Room Temperature Stable (RTS) DNase only when RNA were prepared for quantitative (Q)-RT PCR (Mobio). RNA gel blot analyses were performed as indicated in (52 (link)). Gene-specific probes were amplified by polymerase chain reaction (PCR) using primers indicated in Supplementary Table S1 and radiolabeled with the Prime A Gene labeling kit (Promega) following the recommendations of the manufacturer. Quantitative RT-PCR was performed as previously explained in (52 (link)), using the same set of primers.
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10

RNA Extraction and Transcriptional Analysis

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Tissues were ground in liquid nitrogen and RNA was extracted with Trizol following manufacturer's protocol (Invitrogen™). RNA was further extracted with phenol-chloroform pH 4.3. Five microgram of Turbo DNase (Thermo Fisher) treated RNAs were used for Superscript IV reverse transcription with random hexamers. The resulting cDNA was diluted 20-fold for qPCR reaction. ACT2 (AT3G18780) and TIP41 (AT1G13440) were used as reference genes. For rRNA gel blotting, 0.5–1 μg of RNA (10 μg for other transcripts) was fractionated on 1.2% agarose–1% formaldehyde gel and blotted as described (29 ). Gene PCR products of 200–300 bp were labelled with 32P-dCTP following the prime-a-gene labeling kit instructions (Promega) and used as probes (Supplementary Data Set 2). Results were visualized on an Amersham Typhoon imager and data quantification was performed with ImageJ.
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