Total RNA was extracted from whole blood and subjected to DNAse treatment prior to cDNA synthesis using SuperScript III (Thermo Scientific) and a 1:1 ratio of Oligo dT and random hexamer primers according to the manufacturer’s specification. Exon spanning gene specific primers were used to amplify the regions of interest, which spanned exon 12–16. Specific primer sequences and thermal cycling conditions are available upon request. RT-PCR products were subsequently loaded on 2% Agarose gels and run for 2h at 70V. All visible bands of each affected individual and each parent were gel extracted using the Qiagen MinElute Gel Extraction kit and sub-cloned using the Zero Blunt TOPO PCR Cloning kit (Thermo Scientific) according to the manufacturers’ protocols. A total of 50 clones per individual were randomly selected and sequenced using M13 primers. Sequence traces were aligned to the mRNA reference sequence NM_025082.3 and compared to available transcripts in both RefSeq [37 (link)] and Ensemble [19 (link)].
Minelute gel extraction kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The MinElute Gel Extraction Kit is a tool for the purification of DNA fragments from agarose gels. It allows for the efficient recovery of DNA from gel slices, enabling further downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Zero blunt topo pcr cloning kit, by Thermo Fisher Scientific (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Fastdigest dpni, by Thermo Fisher Scientific (1 mentions)
Pcdna3.1 mammalian expression vector, by Thermo Fisher Scientific (1 mentions)
Qiaamp dna mini kit, by Qiagen (1 mentions)
Quant it dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Minelute pcr purification kit, by Qiagen (1 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Klenow fragment, by New England Biolabs (1 mentions)
T4 polynucleotide kinase, by New England Biolabs (1 mentions)
T4 dna polymerase, by New England Biolabs (1 mentions)
Pmd18 t vector, by Takara Bio (1 mentions)
Ez dna methylation kit, by Zymo Research (1 mentions)
Purelink miniprep kit, by Thermo Fisher Scientific (1 mentions)
Abi prism 7700 sequence detection, by Thermo Fisher Scientific (1 mentions)
Methyl primer express software version 1, by Thermo Fisher Scientific (1 mentions)
5 protocols using minelute gel extraction kit
1
Comprehensive Transcript Analysis of Exon 12-16
2
Cassette Amplification and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The DICE-EPv2.0 and PTKmChR cassettes were amplified from a common vector containing the PuroRΔTK fusion gene (from Addgene # 22733) followed by a P2A ribosomal skipping element and mCherry followed by the rabbit beta-globin terminator. This cassette was under the control of the mouse phosphoglycerol kinase (PGK) promoter. Cassettes from the pKER series were amplified from their respective plasmid vectors once assembled. Cassettes were amplified via PCR with Q5 high-fidelity polymerase and either subjected to gel electrophoresis, excised, and purified with the MinElute Gel Extraction Kit or pooled, column-purified, digested with FastDigest DpnI (Thermo Fisher Scientific), and purified by chromatography. For the DICE-EPv2.0 cassettes, the phiC31 and Bxb1 attP sites were included in the primers used for amplification. All primers used for cassette amplification appear in Supplementary Table S3. Further details of construction, amplification, and purification of the cassettes appear in the Supplementary Materials and Methods. Phosphorothioate bonds were incorporated into primers during synthesis by the manufacturer (IDT).
3
Cloning and Expression of Estrogen Receptor Alpha Isoforms
4
Genomic DNA Extraction and Illumina Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA was extracted from 1 ml of the isolate MET suspension using a QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The concentration and quality of the extracted DNA were determined using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and gel electrophoresis. The DNA was mechanically fragmented by ultrasonic lysis and then gel size-selected for ~500 base pairs (bp) fragment size. Illumina fragment libraries were constructed using Illumina paired-end DNA sample prep kit v1 with some modifications30 (link). The overhangs from the fragmentation were converted into blunt ends using the T4 DNA polymerase, Klenow Fragment, and T4 Polynucleotide Kinase (New England Biolabs, MA, USA). Sequencing adapters were ligated to the ends of the end-repaired DNA fragments. All reaction clean-ups were performed using a MinElute PCR purification kit (Qiagen, Hilden, Germany). Size-selected DNA was purified with a Qiagen MinElute gel extraction kit and quantified using the Quant-it dsDNA HS assay kit (Invitrogen). Subsequently, the constructed DNA libraries was used for paired-end sequencing using an Illumina Hiseq 2500 sequencing platform (Biomarker, Beijing, China)31 (link).
5
Bisulfite Sequencing of Differentially Methylated Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The methylation levels of the selected differentially methylated genes, namely regulating synaptic membrane exocytosis 2 (Rims2), harvey rat sarcoma virus oncogene (Hras1), thymoma viral proto-oncogene 1 (Akt1), and kirsten rat sarcoma virus oncogene homolog (Kras), were determined by BSP. Briefly, 1 microgram of DNA was treated with the EZ DNA Methylation Kit (Zymo Research, Hiss Diagnostics, Germany). The modified DNA was then amplified by PCR with primers designed with the Methyl Primer Express software version 1.0 (Applied Biosystems, Foster City, CA, USA, S3 Table ), which corresponded to regions on the microarrays. PCR products were purified using the MinElute Gel Extraction Kit (Invitrogen, Carlsbad, CA, USA) and cloned into the pMD18-T Vector (Takara). The plasmids were purified using the PureLink Miniprep Kit (Invitrogen, Thermo Scientific Inc, Waltham, MA, USA). The positive clones were confirmed by PCR, and no fewer than 10 clones were randomly selected for each mouse for sequencing using an automatic sequencer (ABI Prism 7700 Sequence Detection, Applied Biosystems, Foster City, CA, USA). Sequencing results were analyzed using QUMA (http://quma.cdb.riken.jp/top/quma_main.html ) [23 (link)].
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!