DNA from P. infestans isolates (US-6, US-8, US-11, US-22, US-23, and US-24) was used for the PCR amplification of a 1004-bp ITS2 fragment. In a 50-μL reaction mixture containing 6 ng of genomic DNA, 1X Taq PCR buffer, 4.0 mM of MgCl2+, 0.03 U/μL of SurePRIME Taq polymerase (MP Biomedicals, Solon, OH, USA), 0.4 mM of dNTPs (New England Biolabs Inc., Ipswich, MA, USA), and 200 nM each of primers PITSstdF and PITSstdR (Table 1 ), the following PCR conditions were used: 95°C for 15 min, followed by 40 cycles at 94°C for 15 s, 58°C for 45 s, and 72°C for 1 min, and a final extension at 72°C for 10 min. The PCR fragments were purified using the Nucleospin PCR clean-up kit (Macherey-Nagel GmbH & Co., Düren, Germany) according to the manufacturer’s recommendations. The purified products were quantified using a 2100 Bioanalyzer instrument (Agilent Technologies, Santa Clara, CA, USA). The DNA was analyzed according to the manufacturer’s instructions and converted into copy numbers, calculated as copy number = [(concentration of amplicon in g/μL)/(1004 bp × 660 g/mole) × (6.022 × 1023)]. This DNA was then used for optimizing the TaqMan qPCR assay and generating the ITS2 copy number standard curve.
Nucleospin pcr clean up kit
Manufactured by Macherey-Nagel
Sourced in Germany
The NucleoSpin PCR Clean-up kit is a laboratory equipment product designed for the purification of DNA fragments from PCR reactions, enzymatic reactions, or other sources. The kit utilizes silica-membrane technology to efficiently remove contaminants, primers, nucleotides, and salts from the DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Smartscribe reverse transcriptase, by Takara Bio (3 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (3 mentions)
Rna micro kit, by Qiagen (3 mentions)
Ampure xp bead based clean up, by Beckman Coulter (3 mentions)
Hifi dna assembly kit, by New England Biolabs (3 mentions)
T4 ligase, by New England Biolabs (3 mentions)
Pfastbac1, by Thermo Fisher Scientific (3 mentions)
T4 ligase buffer, by New England Biolabs (3 mentions)
T4 polynucleotide kinase, by New England Biolabs (3 mentions)
Concanavalin a coated magnetic beads, by Bangs Laboratories (2 mentions)
Anti h3k27me3, by Active Motif (2 mentions)
Kasumi 1 cell line, by American Type Culture Collection (2 mentions)
Concanavalin a beads, by American Type Culture Collection (2 mentions)
Ez dna methylation kit, by Zymo Research (2 mentions)
Dpni, by New England Biolabs (2 mentions)
25u t4 dna polymerase, by New England Biolabs (2 mentions)
E coli dh5α, by Thermo Fisher Scientific (2 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Miseq platform, by Illumina (2 mentions)
2100 bioanalyzer instrument, by Agilent Technologies (1 mentions)
33 protocols using nucleospin pcr clean up kit
1
Quantitative PCR Assay for P. infestans
2
CUT&RUN Profiling of H3K27me3 Chromatin Marks
3
CUT&RUN Profiling of H3K27me3 Chromatin Marks
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CUT&RUN experiments were carried out as previously described (Skene et al., 2018 (link)) with the following modifications: 1–2.5×105 cells were isolated by FACS as described in sections above, bound to Concanavalin A coated magnetic beads (Bangs Laboratories), and permeabilized with 0.025% (wt/vol) digitonin. Permeabilized cells were incubated overnight at 4°C with 5ug of anti-H3K27me3 (Active Motif) and then washed before incubating with protein A-MNase fusion protein (a gift from S. Henikoff) for 15 minutes at room temperature. After washing, cells were incubated in CaCl2 to induce MNase cleavage activity for 30 minutes at 0°C. The reaction was stopped with 2xSTOP buffer (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 μg/mL RNase A, 50 μg/mL glycogen, and 2pg/mL of yeast spike-in DNA). Histone-DNA complexes were isolated from insoluble nuclear chromatin by centrifugation and DNA was extracted with a NucleoSpin PCR Clean-up kit (Macherey-Nagel). For CUT&RUN quantitative PCR, human Kasumi-1 cell line (ATCC CRL-2724) were added before binding the cells to Concanavalin A beads for internal standard instead of yeast spike-in DNA.
4
T-cell Receptor Profiling Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
T-cell receptor analysis was performed as described previously (54 (link)), with minor modifications. Briefly, mRNA was isolated with the RNA microkit (Qiagen) according to manufacturer’s protocol. Isolated mRNA was used for cDNA synthesis with 5’RACE template switch technology to introduce a universal primer binding site, and unique molecular identifiers (UMI’s) were added at the 5’ end of the cDNA molecules using the SMARTScribe Reverse Transcriptase (TaKaRa). cDNA synthesis was followed by an AMPure XP bead-based cleanup (Beckman Coulter). Purified cDNA molecules were amplified in two subsequent PCR steps using the Q5® High-Fidelity DNA Polymerase (New England BioLabs), with an AMPure XP bead-based cleanup in between. PCR products were size selected on gel and purified using the Nucleospin PCR cleanup kit (Machery-Nagel). The PCR products were sequenced via Illumina MiSeq paired end using 2x250 bp sequencing.
5
Reconstitution and Characterization of Recombinant HCMV
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BAC DNA was isolated using the NucleoBond Xtra Midi Kit (Macherey-Nagel, Düren, Germany) and transfected into HFFs in 6-well plates by calcium phosphate transfection (MBS Transfection Kit; Agilent, Waldbronn, Germany), as described in the Supplementary Protocol. To monitor the process of virus reconstitution, a portion of the culture (1 × 10 5 cells) was removed when cultures were passaged, seeded into 24-well plates and stained for viral IE-antigens 3-4 h after adherence. Cells were counted, and the percentage of IE-positive cells was calculated.
To assess individual virus passages for the absence of the BAC vector cassette and the presence of an intact UL74 ORF, DNA from supernatants was purified using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany), and PCR was performed using chloramphenicol resistance gene-(CAM) or UL74-specific primers. The resulting UL74 amplification products were purified with the NucleoSpin PCR Clean-up Kit (Macherey-Nagel) and checked by Sanger sequencing (GATC, Konstanz, Germany).
To assess individual virus passages for the absence of the BAC vector cassette and the presence of an intact UL74 ORF, DNA from supernatants was purified using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany), and PCR was performed using chloramphenicol resistance gene-(CAM) or UL74-specific primers. The resulting UL74 amplification products were purified with the NucleoSpin PCR Clean-up Kit (Macherey-Nagel) and checked by Sanger sequencing (GATC, Konstanz, Germany).
6
TCRβ Sequencing and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TCRβ analysis was performed as described previously (Shugay et al., 2014 (link)), with minor modifications. Briefly, mRNA was isolated with the RNA microkit (Qiagen) according to the manufacturer's protocol. Isolated mRNA was used for cDNA synthesis with 5′RACE template switch technology to introduce a universal primer binding site, and unique molecular identifiers (UMIs) were added at the 5′ end of the cDNA molecules using the SMARTScribe Reverse Transcriptase (TaKaRa). cDNA synthesis was followed by an AMPure XP bead-based clean-up (Beckman Coulter). Purified cDNA molecules were amplified in two subsequent PCR steps (25 cycles in PCR1 and 20 cycles in PCR2) using the Q5® High-Fidelity DNA Polymerase (New England BioLabs), with an AMPure XP bead-based clean-up in between. PCR products were size-selected on gel and purified using the Nucleospin PCR clean-up kit (Machery-Nagel). The PCR products were sequenced via Illumina MiSeq paired end 2x250 nucleotide (nt) sequencing.
7
Quantification of DNA Methylation via Bisulfite Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bisulfite conversion was done using the EZ DNA Methylation kit from Zymo Research as described before (13 (link)). We converted 200 ng of DNA at 50°C for 12 h from each cell line after 30 days of culturing with ABA. We used primer oVIN-2209 and oVIN-2211 to amplify the converted DNA (Supplementary Material, Table S4 ). The products were then purified using the NucleoSpin PCR Clean-up kit (Macherey-Nagel). We then performed 2 × 250 bp paired-end MiSeq sequencing (Illumina). The sequencing primers are found in Supplementary Material, Table S4 . We processed the reads with TrimGalore (github.com/FelixKrueger/TrimGalore ) using -q 20 —length 20 –paired. We aligned the reads using QuasR (62 (link)) to the GFP transgene sequence. We extracted the methylation levels for each CpG in the amplicon with the qMeth() function in QuasR. We calculated the CpG methylation frequencies by dividing the frequency of methylated CpGs by the total number of CpG and expressed it as a percentage.
8
Expression and Purification of PEDV Spike Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein sequence of Porcine epidemic diarrhea virus (PEDV) USA/Colorado/2013 spike ectodomain 1-1322 with a C-terminal foldon, TEV site, His-tag and Strep-tag was codon optimized for mammalian expression (Genscript) and inserted into pFastBac1 (Thermo Fisher, Cat. #10360014) using EcoRI and XbaI restriction sites (Genscript). The insert was further cloned into pcDNA3.4 using the HiFi DNA Assembly Kit (New England Biolabs) and PCR products (Phusion polymerase, Thermo Fisher, Cat. #F530L) for spike (PEDV fwd and rvs) and for pcDNA3.4 (pcDNA34 fwd and rvs).
The Asn264Asp in spike-pcDNA3.4 substitution and 34-230 deletion spike (based on genbank sequence AMK69964) in spike-pFastBac1 mutants were made by Phusion PCR of the parent templates using N264D fwd and rvs or PEDVS0033 rvs and PEDVS0231 fwd primers. The resulting PCR products were purified with NucleoSpin PCR cleanup kit (Machery Nagel, Cat. #740609.50) to which was added 10X PNK buffer, 1 µL T4 polynucleotide kinase and 1 µL DpnI (New England Biolabs, Cats. #M0201S and R0176S) and incubated at 37˚C for 1 hour. 3µL of this reaction, 1µL of 10X T4 ligase buffer, 1µL of T4 ligase (New England Biolabs, Cat. #M0202S) and 5µL of water were incubated at room temperature for 1 hour. The ligated DNA was then transformed into DH5a E. coli (Thermo Fisher, Cat. #18258012) and selected on LB broth agar with 100µg/mL ampicillin.
The Asn264Asp in spike-pcDNA3.4 substitution and 34-230 deletion spike (based on genbank sequence AMK69964) in spike-pFastBac1 mutants were made by Phusion PCR of the parent templates using N264D fwd and rvs or PEDVS0033 rvs and PEDVS0231 fwd primers. The resulting PCR products were purified with NucleoSpin PCR cleanup kit (Machery Nagel, Cat. #740609.50) to which was added 10X PNK buffer, 1 µL T4 polynucleotide kinase and 1 µL DpnI (New England Biolabs, Cats. #M0201S and R0176S) and incubated at 37˚C for 1 hour. 3µL of this reaction, 1µL of 10X T4 ligase buffer, 1µL of T4 ligase (New England Biolabs, Cat. #M0202S) and 5µL of water were incubated at room temperature for 1 hour. The ligated DNA was then transformed into DH5a E. coli (Thermo Fisher, Cat. #18258012) and selected on LB broth agar with 100µg/mL ampicillin.
9
In Vitro Synthesis and Labeling of Pre-miR-181a-1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Pre-miR-181a-1 DNA template was obtained by two oligos (sequence below) after annealing and elongation at 12°C using 25U T4 DNA polymerase (NEB). The DNA template was purified with NucleoSpin PCR Clean-up kit (Macherey-Nagel). Pre-miR-181a-1 in vitro synthesis was performed using the T7 MEGAshortscript kit (Ambion) from 1 µg purified DNA and following manufacturer's instructions, including template removal by DNase TURBO digestion. An amount of 5 µg of pre-miR-181a-1 was labeled with cy3 using the Nucleic Acid Labeling kit (Mirus) following the manufacturer's instructions.
Pre-miR-181a-1_T7_Fw: 5′-TAATACGACTCACTATAGAACATTCAACGCTGTCGGTGAGTTTGGTATCTAAAGGC-3′
Pre-miR-181a-1_Rv: 5′-TGTACAGTCAACGATCGATGGTTTGCCTTTAGATACCAAACTCACCG-3′
Pre-miR-181a-1_T7_Fw: 5′-TAATACGACTCACTATAGAACATTCAACGCTGTCGGTGAGTTTGGTATCTAAAGGC-3′
Pre-miR-181a-1_Rv: 5′-TGTACAGTCAACGATCGATGGTTTGCCTTTAGATACCAAACTCACCG-3′
10
Axolotl TERT Partial Sequence Cloning
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Contigs with sequence identity to human TERT were searched in the Ambystoma Mexicanum V4.0 (contigs) database, via its website, Sal-SiteTM [32 ]. Five Contigs (contig282000, contig 346319, FUQAVB301DCODH, GFW9XCP01AHQ6K, and GHXAJEM01EV71Q) were assembled and used for Primer derivation. The primers’ sequences were (5′-AAATGGTTTGCGCCCGATAGTC-3′ and 5′-ATAAAGGCATGGTAGCTAAGCCACTG-3′). Axolotl embryonic cDNA isolated for other projects was obtained from laboratory stock for PCR establishment. PCR was performed utilizing the Advantage® 2 PCR Kit (Takara Bio Inc., Kusatsu, Japan). The amplificate was purified by Nucleo Spin® PCR Clean up Kit (Macherey Nagel) and cloned into pUC118 vector [33 (link)] using the Mighty Cloning Reagent Set (Blunt end) (Takara Bio Inc., Kusatsu, Japan) according to the manufacturer’s protocol. The cloned DNA was sequenced by Sanger sequencing using an M13 primer (GATC Services, Eurofins Genomics, Ebersberg, Germany). A BLASTn [34 ] search in the Nucleotide Collection (nr/nt) database exhibited a high concordance with other TERT nucleotide sequences, so this cloned fragment was supposed to be part of Amex Tert.
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!