Clone libraries were built with PAH-RHDα GN and GP amplicons obtained by qPCR on cDNA fractions from soil after 10 days incubation. qPCR reactions from triplicate microcosms were pooled. Amplicons were purified using the Cycle Pure Kit (Omega Bio-Tek) and cloned in the pGEM-T Vector System (Promega). Plasmids from ampicillin-resistant clones were purified using the E-Z 96 FastFilter Plasmid DNA kit (Omega Bio-Tek) and sequenced in one direction using the M13uni-21 forward primer. Sequences were edited in SeqTrace (Stucky, 2012 (link)) to clip PAH-RHDα primers and compared to the NCBI nr database using blastn (Madden, 2002 ) to retrieve closely related sequences. Alignments for PAH-RHDα GN and GP produced using MUSCLE (Edgar, 2004 (link)) were manually edited in MEGA6 (Tamura et al., 2013 (link)), and final versions comprising respectively 251 and 205 ungapped positions were used to infer maximum-likelihood phylogenetic trees with 100 resamplings.
Cycle pure kit
Manufactured by Omega Bio-Tek
Sourced in United States, China
The Cycle Pure Kit is a lab equipment product developed by Omega Bio-Tek. It is designed to perform purification of PCR amplicons and other DNA fragments. The kit utilizes a unique magnetic bead-based technology to efficiently capture and concentrate the target DNA, allowing for the removal of unwanted reagents, primers, and primer dimers.
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Lab products found in correlation
Gel extraction kit, by Omega Bio-Tek (4 mentions)
Pgem t easy vector, by Promega (3 mentions)
Smart race kit, by Takara Bio (3 mentions)
Pgem t vector system, by Promega (2 mentions)
Pgem easy vector system 2, by Promega (2 mentions)
Bigdye terminator v3.1 cycle sequencing kit, by Thermo Fisher Scientific (2 mentions)
Smart race cdna amplification kit, by BD (2 mentions)
Abi 3100 automated capillary sequencer, by Thermo Fisher Scientific (2 mentions)
Pgem t vector, by Promega (2 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (2 mentions)
Cytation 5 plate reader, by Agilent Technologies (2 mentions)
Accuzyme dna polymerase, by Meridian Bioscience (2 mentions)
Plasmid mini kit 1, by Omega Bio-Tek (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Bamhi, by Takara Bio (1 mentions)
Bigdye terminator v3.1 kit, by Thermo Fisher Scientific (1 mentions)
T7 ribomax express rnai system, by Promega (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
47 protocols using cycle pure kit
1
Metagenomic Analysis of PAH-RHDα Genes
2
Host Size and Microsporidian Infection in Asellus aquaticus
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To analyse the relationship between host size and parasite infection, the length of each A. aquaticus individual was measured according to images taken with a stereo microscope equipped with a camera (moticam 2300, Motic®) that was calibrated with a scaled slide. The animals were cut approximately in the sagittal plane and the intestine was removed to avoid contamination with gut content. The DNA was extracted from the remaining tissue following the procedure described in Grabner et al. (2015 (link)). Detection of microsporidians was conducted using the universal microsporidian primers V1 (5′-CACCAGGTTGATTCTGCCTGAC-3′) (Zhu et al., 1993 (link)) and mic-uni3R (5′-ATTACCGCGGMTGCTGGCAC-3′) (Weigand et al., 2016 ). PCR thermal profiles and reaction volumes were conducted as described in Weigand et al. (2016 ). PCR products were purified using an E.Z.N.A. Cycle Pure kit (Omega Bio-Tek) and sent for Sanger sequencing (Microsynth Seqlab) using primer V1. Raw sequences were quality-checked using Geneious v2022.0.1 (Biomatters).
3
Preparation of Nicked Plasmid DNA
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Plasmid pET-30a (+) DNA can be nicked by GVE2 HNH endonuclease (Zhang et al. 2016) . Thus, the nicked plasmid pET-30a (+) DNA was prepared in the DNA nicking reaction containing 200 ng plasmid pET-30a (+) DNA, 20 mM Tris-HCl (pH 8.0), 5.0 mM DTT, 200 nM GVE2 HNH endonuclease, 1.0 mM MgCl 2 and 10 % glycerol. The reactions were kept at 60 o C for 3 min to ensure maximum yield of the nicked DNA. The nicked DNA product was purified by using a Cycle Pure Kit (Omega Bio-Tek, Norcross, GA, USA).
As shown in Table 1, the Cy3-labeled nucleotide duplex (*p22-37/t59) with a single nick was prepared by annealing the Cy3-labeled nucleotide (*p22) and the phosphorylated nucleotide (p37) with the complementary nucleotide (t59) in a buffer containing 20 mM Tris-Cl (pH 8.0) and 100 mM NaCl. The mixture was heated at 100 o C for 5 min and cooled slowly at least 4 hours to room temperature.
As shown in Table 1, the Cy3-labeled nucleotide duplex (*p22-37/t59) with a single nick was prepared by annealing the Cy3-labeled nucleotide (*p22) and the phosphorylated nucleotide (p37) with the complementary nucleotide (t59) in a buffer containing 20 mM Tris-Cl (pH 8.0) and 100 mM NaCl. The mixture was heated at 100 o C for 5 min and cooled slowly at least 4 hours to room temperature.
4
Chromatin Immunoprecipitation in Adipocytes
5
Chromatin Immunoprecipitation in Adipocytes
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Cultured inguinal adipocyte of both wild-type and mutant mice was cross-linked with 1% formaldehyde in DMEM medium for 10 minutes at room temperature followed by the addition of 125 mM glycine for 5 min at room temperature, after which cells were washed 1 time with ice-cold PBS and scraped into SDS lysis buffer. The cells were further sonicated and diluted for immunoprecipitation with the indicated antibodies. The immunoprecipitates were eluted and reverse cross-linked overnight at 65 °C. DNA fragments were purified using the Cycle Pure kit (Omega Bio-Tek), and qPCR was performed with primers listed in Supplementary Table 1 . Genomic DNA was extracted from adipose tissue and amplified with primers listed in supplementary Table 1 .
6
Molecular Characterization of SfMNPV Isolates
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Virus DNA samples were purified from OBs, as previously described [23 (link)], and digested with PstI and BamHI (Takara Bio, Inc., Otsu, Japan). Restriction endonuclease (REN) profiles were visually analyzed using the Syngene G: BOX Chemi imaging system (Syngene, Cambridge, United Kingdom). A DNA marker (Trans Gene Biotech, Beijing, China) was used to indicate the molecular size of the DNA fragments.
The amplification of partial polh, lef-8, and lef-9 genes were performed using degenerate baculoviruses primer pairs (prPH-1, prPH-2, prL8-1, prL8-2, prL9-1, and prL9-2) and same reaction conditions as mentioned in Jehle et al. [24 (link)]. The PCR products were purified by using the Cycle-Pure Kit (OMEGA bio-tek, Norcross, United States), and were sequenced by automatic sequencing (Sangon Biotech, Shanghai, China). Partial polh, lef-8, and lef-9 sequences were submitted to the GenBank with the accession numbers MW115954, MW115952, and MW115953. The Kimura 2-parameter (K-2-P) distances of lef-8, lef-9, and polh fragments among SfMNPV-Hub and other seven sequenced SfMNPV isolates were calculated using Kimura 2-parameter model by MEGA 7.0 [25 (link)].
The amplification of partial polh, lef-8, and lef-9 genes were performed using degenerate baculoviruses primer pairs (prPH-1, prPH-2, prL8-1, prL8-2, prL9-1, and prL9-2) and same reaction conditions as mentioned in Jehle et al. [24 (link)]. The PCR products were purified by using the Cycle-Pure Kit (OMEGA bio-tek, Norcross, United States), and were sequenced by automatic sequencing (Sangon Biotech, Shanghai, China). Partial polh, lef-8, and lef-9 sequences were submitted to the GenBank with the accession numbers MW115954, MW115952, and MW115953. The Kimura 2-parameter (K-2-P) distances of lef-8, lef-9, and polh fragments among SfMNPV-Hub and other seven sequenced SfMNPV isolates were calculated using Kimura 2-parameter model by MEGA 7.0 [25 (link)].
7
TP53 Gene Amplification and Sequencing
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A PCR amplification instrument was utilized to amplify the target fragment of TP53. Amplification cycle conditions were as follows: 95°C for 5 min followed by 40 cycles of 95°C for 1 min, 53°C for 1 min, 72°C for 1 min, and a final elongation at 72°C for 10 min. The samples were purified using a Cycle Pure Kit (D6492-02, Omega Biotek, USA), sequenced with Big Dye Terminator v3.1 kit (Thermo Fisher Scientific, USA), then purified. Finally, sequencing analysis was performed by ABI 3500 gene sequencer.
8
RNAi dsRNA Synthesis and Purification
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Following the manufacturer’s protocol, the corresponding dsRNA was synthesized using the T7 RiboMAX Express RNAi system of the dsRNA synthesis kit (Promega, Madison, WI, USA). Bta06987fragments for dsRNA synthesis were amplified by specific primers containing the T7 promoter sequence (Table 1 ). The primers are designed by Primer 5 basing on the sequence of Bta06987 (Table S1 ). After purification using the cycle pure kit (Omega Biotek Inc., GA, USA), the PCR product was mixed with T7 Express Enzyme Mix and RiboMAX™ Express T7 2 × Buffer at a specific proportion. The mixture was incubated at 37 °C for 2–6 h and at 70 °C for 10 min, and the mixture was then transferred for incubation at room temperature for 20 min to promote dsRNA formation. To finish, we added DNase and RNase A into dsRNA to remove DNA template and single-stranded RNA, respectively. The quality of dsRNA was determined on a 2% agarose gel and the quantity of dsRNA was measured by Nanodrop 2000.
9
High-throughput sequencing of genetic diversity
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One set of 85 EcoRI-HpaII ligated samples were pooled and purified in one tube, and another set of 85 EcoRI-MspI ligated samples were pooled and purified in another tube using an E.Z.N.A. Cycle-Pure Kit (Omega Bio-tek, Inc., US, D6492). Restriction fragments from each library were then amplified in 50 μL volumes containing 10 μL of pooled DNA fragments, NEB 2x Taq Master Mix (New England Biolabs), and 20 μM each of the primer pairs. The EcoRI-HpaII primer pair sequences were 5′-TAGCTCGTAGACACCGTCAG-3′ and 5′-GTCATGCCTCATCTCACCGG-3′, and the EcoRI-MspI primer pair sequences were 5′-TAGCTCGTAGACACCGTCAG-3′ and 5′-GTCATGCCTCATTAGTCCGG-3′. PCR was performed at 95°C (30s), followed by 23 cycles consisting of 95°C (30 sec), 55°C (30 sec), and 68°C (30 sec), and a final step at 72°C (5 min). The samples were then run on a 2% agarose gel (Sigma). PCR products ranging from 250 bp to 500 bp in size were isolated using an E.Z.N.A. Gel Extraction Kit (Omega Bio-tek Inc., US, D2500-1). After EcoRI-MspI 85-plex library and EcoRI-HpaII 85-plex library had been constructed, equal amount of the two libraries were combined into one 85-plex library. Then paired-end sequencing was performed in one lane using an Illumina HiSeq 2500 (Illumina Inc., San Diego, CA) by Shanghai South Gene Technology Co., Ltd.
10
ChIP-qPCR for p300 in EB
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Following EB formation, the cells were fixed with formaldehyde on day 4 of differentiation, sonicated with a Bioruptor® and the chromatin was immunoprecipitated with a p300 antibody as described previously22 (link)57 (link). A sample of 5% of total chromatin was used as the input control. The antibody against p300 was from Santa Cruz Biotechnology and the negative control IgG antiserum was from Zymed Laboratories Inc. The immunoprecipitated DNA was purified using the Omega Bio-tek Cycle Pure Kit, and the purified DNA samples were analyzed using SYBR Green Real-Time PCR. Primer pairs used for PCR amplification were described previously14 (link).
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