To determine the mutational status of the TERT promoter, we used a nested PCR strategy with our FFPE patient samples. Genomic DNA in this region is 78% GC rich and it was necessary to use PCR additives to amplify the region. The primers were designed and tested in silico using PRIMER3 software. The first round PCR was performed with primers hTERTF x hTERTR under the following conditions: initial denaturation at 98°C for 30 seconds followed by 36 rounds of 98°C denaturation for 10 s and combined annealing and extension at 72°C for 25 sec. We employed the Q5 high fidelity DNA polymerase (New England Biolabs; NEB) and the supplied 5x PCR buffer and GC enhancer (NEB) to amplify a 474 bp region. The second round of PCR was initiated with 0.2 ul template taken directly from the first round using primers TERT5 F x TERT5 R again with the addition of the GC enhancer and the same conditions increased to 40 cycles. PCR reactions were run on a 2% TBE agarose gel and the expected 193 bp product was excised and gel purified using a Monarch Gel Extraction Kit (NEB) following the product protocol. The purified fragments were Sanger sequenced at the London Regional Genomics Facility with betaine (1M) added to the reaction. Chromatograms were aligned and compared against the TERT sequence.
Monarch gel extraction kit
Manufactured by New England Biolabs
Sourced in United States
The Monarch Gel Extraction Kit is a laboratory product designed to efficiently extract and purify DNA fragments from agarose gels. It provides a simple and reliable method for DNA recovery from gel slices.
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Lab products found in correlation
Trizol, by Thermo Fisher Scientific (3 mentions)
Trans1 t1 clone vector, by Transgene (3 mentions)
Primer premier 5, by Premier Biosoft (2 mentions)
Nucleospin tissue, by Macherey-Nagel (2 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)
Qiaprep spin miniprep kit, by Qiagen (1 mentions)
Blunt ta ligase master mix, by New England Biolabs (1 mentions)
Ssiii one step rt pcr kit with platinum taq, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Hiseq 2500 instrument, by Illumina (1 mentions)
Bioanalyzer high sensitivity dna kit, by Agilent Technologies (1 mentions)
Monarch pcr cleanup kit, by New England Biolabs (1 mentions)
Hiseq rapid sbs kit v2, by Illumina (1 mentions)
Dna 1000 kit, by Agilent Technologies (1 mentions)
Abi 3130 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Bigdye v1.1 kit, by Thermo Fisher Scientific (1 mentions)
Tempus spin rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Tempus blood rna tube, by Thermo Fisher Scientific (1 mentions)
Superscript 4 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Onetaq 2x master mix, by New England Biolabs (1 mentions)
23 protocols using monarch gel extraction kit
1
TERT Promoter Mutation Detection
2
Viral Genome Amplification and Cloning
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Total RNA was isolated from C6/36 cells infected with PaRV, PCV, BinJV or HVV at 5dpi and from KRBV-positive mosquitoes, and RT-PCR was used to amplify the fragment of the viral genome containing 3’UTR and the small terminal part of the NS5 gene. RT-PCR was performed using SSIII One-Step RT-PCR Kit with Platinum Taq (Invitrogen, USA) according to the manufacturer’s recommendations. PCR primers (Supplementary Table 2 ) were 5’-phosphorylated, and forward primers were designed to incorporate T7 promoter at the 5’-end of the amplicons. The cycling conditions were 15 min at 60 °C, 2 min at 95 °C; 35 cycles of 15 s at 95 °C, 30 s at 55 °C, 40 sec at 68 °C; and a final extension for 5 min at 68 °C. PCR products were then separated in 2% agarose gel, purified using Monarch Gel Extraction Kit (NEB, USA) and ligated with SmaI-digested and dephosphorylated pUC19 vector using Blunt/TA Ligase Mastermix (NEB, USA). Ligation was performed overnight at 16 °C, and products were transformed into NEB5α chemically competent cells (NEB, USA) and plasmids were isolated using QIAprep Spin Miniprep Kit (Qiagen, Germany).
3
Cloning and Sequencing of MRLC-sqh
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Total RNA was extracted from 3-day-old adult O. communa samples using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. First strand cDNA synthesis was performed using 1.0 µg total RNA and the One-step Reagent Kit with gDNA Eraser (TransGen Biotech, Beijing, China). Subsequently, according to the sequence obtained from the previous transcriptome data (shown in the Supplementary Materials ), a pair of specific primers were designed using Primer Premier 5 (PREMIER Biosoft International, Palo Alto, CA, USA; Table 1 ). They were used to amplify the MRLC-sqh sequence using the cDNA as a template. The amplification product was then purified using the Monarch Gel Extraction kit (NEB, Ipswitch, MA, USA) and cloned into a Trans1-T1 clone vector (TransGen Biotech) and sequenced [33 (link)].
4
Small RNA Library Preparation and Sequencing
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Equal amounts of RNA of the biological replicates were pooled, and 6.0 ng total RNA were used as starting material for library preparation using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs, USA) in accordance with the manufacturer's instructions. After a pre-amplification, PCR products were purified by applying the Monarch PCR Cleanup Kit (New England Biolabs). Then, cDNA libraries were evaluated via capillary gel electrophoresis using the DNA 1000 Kit and the Bioanalyzer 2100 (Agilent Technologies) according to the manual. A miRNA-specific length of 130–150 base pairs of barcoded cDNA libraries was selected by applying and fractionating 5 ng of pooled cDNA on a 4% agarose gel (MetaPhor, USA). Clean-up of cut gel slices at the appropriate size range was performed using the Monarch Gel Extraction Kit (New England Biolabs) and correct size and molarity were analyzed via capillary gel electrophoresis using the Bioanalyzer DNA High Sensitivity Kit (Agilent Technologies). Finally, 50 cycles of single-end sequencing-by-synthesis reactions were conducted on the HiSeq 2500 instrument (Illumina, USA) with the HiSeq Rapid SBS Kit v2 (Illumina, USA).
5
RNA Isolation and Sequencing Protocol
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RNA was isolated using the Tempus Spin RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA) from peripheral blood taken in Tempus Blood RNA tubes (Thermo Fisher Scientific) according to the manufacturer’s recommendations. First-strand reverse transcription was carried out by SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific). Reverse transcription PCRs (RT-PCRs) amplifying the variant-containing exons along with at least two adjacent exons were designed individually (list of cDNA primers is given in S2 Table ). Amplification products were visualized on 1% agarose gel next to Hyper Ladder 1 kb DNA sizing standard (Bioline, London, UK) and subsequently sequenced by conventional Sanger sequencing method on ABI3130 Genetic Analyzer using the BigDye v.1.1 Kit (Thermo Fisher Scientific). Sequencing was done for the whole RT-PCR product without separation of the respective bands to compare peak intensities of normal and aberrantly spliced products. Where it was necessary to remove the interfering predominant normal alternative splice product, fragments of different sizes were cut out and cleaned from the gel by Monarch Gel Extraction Kit (New England Biolabs, Ipswich, MA) and the purified product was sequenced as above.
6
Confirming SNP Genotyping by Sequencing
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A DNA segment containing each SNP was amplified from 5 randomly chosen samples to be used as a template for sequencing in order to confirm the allelic discrimination performed with Real time PCR. Primers were designed using primer3 tool (https://primer3.ut.ee/ ) using the Ensemble (ENST00000647318.2 ENSG00000167207) NOD2 gene annotation system. The primers were evaluated with the in-silico PCR UCSC Genome Browser tool (https://genome.ucsc.edu/ ) to determine their size and accuracy. The PCR sequences and the size of the amplified primer are described in S3 Table . Samples were prepared to a final volume of 20 μl containing 10 μl of OneTaq® 2X Master Mix (NEW ENGLAND BioLabs Inc), 0,4 μl of each primer (10 μM) and 2 μl of DNA (2,5 ng/ μl) and 7,2 μl of MilliQ water. Amplification conditions were initial denaturation at 95°C for 3m and 40 cycles of denaturation at 95°C for 10s, annealing at 60°C for 30 s and extension 72°C for 30s, with a final extension at 72°C for 2 m. PCR products, used for sequencing, were purified using the Monarch® gel extraction kit from New England BioLabs Inc.
7
Transcriptome Analysis of Mythimna separata
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Various larval instars were collected and flash-frozen in liquid nitrogen for the subsequent transcriptome sequencing, which was performed by Annoroad Gene Technology company (Beijing, China). The M. separata transcriptome database was screened for the TPS sequence, which was then analyzed and compared with the sequences in the NCBI database.
Total RNA was extracted from fourth instar larvae using TRIzol (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1.0 µg total RNA using the ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Shanghai, China). On the basis of the screened sequence, primers MsTPS-F and MsTPS-R (Supplementary Table S1 ) were designed using the Primer Premier 5.0 software for the PCR amplification of the MsTPS sequence. The synthesized cDNA served as the template for the PCR. The PCR amplification product was then purified using the Monarch Gel Extraction kit (NEB, Ipswich, MA, United States), inserted into the Trans1-T1 cloning vector (TransGen Biotech, Beijing, China), and sequenced to confirm its accuracy.
Total RNA was extracted from fourth instar larvae using TRIzol (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1.0 µg total RNA using the ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Shanghai, China). On the basis of the screened sequence, primers MsTPS-F and MsTPS-R (
8
Genomic Analysis of Nectin-1 Gene in Mice
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Genomic DNA of tails collected from mice was extracted using NucleoSpin Tissue (Macherey-Nagel, Düren, Germany). The targeted fragments around the sgRNA targeting site from the extracted
genomic DNA as a part of the nectin-1 gene was amplified with TAKARA Ex Taq (Takara Bio) and the following primers: M-nectin-1_1st_F (5′-GTGGTGCAGGTGAACGACT-3′) and M-nectin-1_1st_R (5′-
CCATCACAGTGAGATTGAGC-3′) as 1st primers pair; M-nectin-1_2nd_F (5′- CTCCATGTATGGCTTCATCG-3′) and M-nectin-1_2nd_R (5′- CAGTGAGATTGAGCTGGCTTT-3′) as 2nd primers. The PCR product was purified
with a Fast Gene Gel/PCR Extraction Kit (Nippon Genetics, Tokyo, Japan) and were additionally purified by agarose gel electrophoresis and Monarch Gel Extraction Kit (New England BioLabs,
Inc., Beverly, MA, USA). The purified PCR products were subsequently sequenced with M-nectin-1_2nd_F primer, using a BigDye Terminator v3.1 Sequencing Kit (Applied Biosystems, Inc., Foster
City, CA, USA).
genomic DNA as a part of the nectin-1 gene was amplified with TAKARA Ex Taq (Takara Bio) and the following primers: M-nectin-1_1st_F (5′-GTGGTGCAGGTGAACGACT-3′) and M-nectin-1_1st_R (5′-
CCATCACAGTGAGATTGAGC-3′) as 1st primers pair; M-nectin-1_2nd_F (5′- CTCCATGTATGGCTTCATCG-3′) and M-nectin-1_2nd_R (5′- CAGTGAGATTGAGCTGGCTTT-3′) as 2nd primers. The PCR product was purified
with a Fast Gene Gel/PCR Extraction Kit (Nippon Genetics, Tokyo, Japan) and were additionally purified by agarose gel electrophoresis and Monarch Gel Extraction Kit (New England BioLabs,
Inc., Beverly, MA, USA). The purified PCR products were subsequently sequenced with M-nectin-1_2nd_F primer, using a BigDye Terminator v3.1 Sequencing Kit (Applied Biosystems, Inc., Foster
City, CA, USA).
9
Rapid Genotyping of Transgenic Models
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A step-by-step protocol is provided in the supplementary Materials and Methods . F0 or F1 animals were genotyped by first extracting genomic DNA from whole embryos at 3-5 dpf using the HOTSHOT method (Meeker et al., 2007 (link)) or the Monarch Genomic DNA Purification Kit (New England Biolabs) to isolate high-molecular weight genomic DNA. PCR was then performed using Onetaq or Q5 (New England Biolabs) with the primer pairs indicated in Table S3 and according to the manufacturer's instructions, typically using 35-40 amplification cycles. For this analysis, locus-specific genotyping primers were located outside of the region used for the homology arms in the HDR template. PCR products were electrophoresed in 1-2% agarose gels. Products from F0 and F1 genotyping were purified with the Monarch Gel Extraction Kit (New England Biolabs) and verified by Sanger sequencing (Source Bioscience) with the PCR primers.
10
Targeted NGS Splicing Profiling
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NGS amplicons were designed to span splicing junctions of interest, with 4 staggered forward primers and a single reverse primer per assay (Supplementary Table 4 ). Sequencing primers were designed with 65 ℃ binding temperatures. Sequencing was performed in two PCR steps, with the first step amplifying the region of interest and the second barcoding the amplicons using Illumina standard adaptors. For some lower TPM genes, additional first-round cycles were used to increase yield. Barcoded wells from the 2nd-round NGS product were pooled by experiment and loaded onto E-gel EX 2% Agarose gels (Invitrogen, G401002) for visualization. Amplicons were purified using the Monarch Gel Extraction Kit (New England Biolabs, T1030) before sequencing using an Illumina MiSeq. Following sequencing, raw reads were analyzed using a custom R script, calculating a percentage by searching for counts of the wildtype and trans-product splicing junctions.
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