Favorprep gel pcr purification kit

Manufactured by Favorgen Biotech

Sourced in Austria

The FavorPrep™ GEL/PCR Purification Kit is a laboratory tool designed for the purification of DNA fragments from agarose gels and the cleanup of PCR (Polymerase Chain Reaction) amplification products. The kit utilizes a silica-membrane-based technology to efficiently capture and purify DNA, removing unwanted components such as primers, nucleotides, and enzymes.

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34 protocols using favorprep gel pcr purification kit

Genomic DNA Isolation and TP53 Gene Sequencing

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Genomic DNA was isolated from Mero-14 cell line using Phase Lock Gel™ (PLG) (VWR International Ltd, UK) according to the manufacturer’s genomic DNA isolation instruction. The entire coding sequence of the TP53 gene (exons 1–11) was amplified using designed primers by Liu and Bodmer [37 (link)].
The standard PCR was performed in a volume of 50 µl containing 200 ng template DNA, 10 pmol of each primer, 25 µl of MyTaq™Red Mix 2X (Bioline, UK) and the appropriate volume of H₂O. PCR programs were then used to amplify the exons: initial denaturation (1 min at 95 °C), followed by 35 cycles of denaturation (95 °C for 15 s), annealing temperature (Tm; 56 °C for 15 s for all exons, except exon 2 Tm: 57 °C), and extension (72 °C for 30 s), and a final extension of 72 °C for 3 min. PCR products were purified using FavorPrep™Gel/PCR Purification Kit (Favorgen, Germany). 10 ng/µl of purified PCR product were sent to Source BioScience (UK) for Sanger sequencing. Sequence alignment was performed using BioEdit software v7.0.5.3. Gene-Bank accession number NG_017013.2 was used as a reference sequence. In addition, sequence that corresponds to the genomic sequence NC_000017.10 was used for exon alignment.

Tian K., Bakker E., Hussain M., Guazzelli A., Alhebshi H., Meysami P., Demonacos C., Schwartz J.M., Mutti L, & Krstic-Demonacos M. (2018). p53 modeling as a route to mesothelioma patients stratification and novel therapeutic identification. Journal of Translational Medicine, 16, 282.

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Quantification of E. canis DNA using ddPCR

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LODs between ddPCR and cPCR were evaluated in sample with known template concentrations.
DNA was extracted from an E. canis infected dog. The 16SrDNA gene was amplified using cPCR with the newly designed primers. The known
16S rDNA copy number sample was prepared using the 16SrDNA amplicons. The amplicons were purified using a FavorPrep™ GEL/PCR Purification Kit
(Favorgen, Ping-Tung, Taiwan), to obtain the known 16S rDNA gene in mole
equivalents. The molecular weight of double stranded 16S rDNA amplicons
was estimated based on the Sequence Manipulation Suite
(https://www.bioinformatics.org/sms2/dna_mw.html), showing 83,901.74 g/mol. The amplicons
concentration was initially adjusted to 1 ng/µl. This concentration was recalculated as
described previously [59 (link)] using these steps:
1 ng/µl = 1×10-983,901.74 mol/µl = 1.19 ×
10⁻¹⁴ × 6.02 × 10²³= 7.175 × 10⁹ molecules/µl
(copies/µl)
Then, the sample was adjusted to 10,000 copies/µl before being ten-fold diluted from
10,000 to 0.01 copies/µl. 2 µl of each diluted amplicon that was used as a template for
ddPCR and cPCR assays. Since one E. canis genome had only one copy of the
16S rDNA gene [65 (link)], the gene
copy number can be directly converted into the number of E. canis.

WICHIANCHOT S., HONGSRICHAN N., MANEERUTTANARUNGROJ C., PINLAOR S., IAMROD K., PURISARN A., DONTHAISONG P., KARANIS P., NIMSUPHAN B, & RUCKSAKEN R. (2022). A newly developed droplet digital PCR for Ehrlichia canis detection: comparisons to conventional PCR and blood smear techniques. The Journal of Veterinary Medical Science, 84(6), 831-840.

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Amplification and Cloning of MYB10 Promoter

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Gene and promoter amplification were performed using MyFiÔ DNA Polymerase (Bioline). FvMYB10-gypsy and Fvb1 FIN12 genomic fragments flanking the deleted region were amplified with the 5Prime PCR Extender System (5Prime) using the provided 103 Tuning Buffer following the manufacturer instructions.
PCR products <8 kb were purified with a FavorPrep GEL/PCR Purification Kit (Favorgen). PCR products >8 kb were precipitated (30% [w/v] PEG8000; 30mN MgCl 2 ). Purified products were cloned into the pGEM-T Easy Vector Systems (Promega) for Sanger sequencing.
Multiple sequence alignment of MYB10 promoter fragments was performed using the Geneious algorithm (Gap open penalty 5 30; Gap extension penalty 5 0; 50 refinement iterations) and used for homology tree construction (Genetic distance model: Jukes-Cantor; Tree build method: Neighbor-Joining; Resampling method: Bootstrap 1000 replicates), both in Geneious 7.1.9 (Kearse et al., 2012) . F. vesca MYB10 pro sequence was chosen as outgroup as it belongs to a different species. Promoter sequence alignment and machine-readable tree files are provided as Supplemental Data Sets 12 and 13.

Castillejo C., Waurich V., Wagner H., Ramos R., Oiza N., Muñoz P., Triviño J.C., Caruana J., Liu Z., Cobo N., Hardigan M.A., Knapp S.J., Vallarino J.G., Osorio S., Martín-Pizarro C., Posé D., Toivainen T., Hytönen T., Oh Y., Barbey C.R., Whitaker V.M., Lee S., Olbricht K., Sánchez-Sevilla J.F, & Amaya I. (2020). Allelic Variation of MYB10 Is the Major Force Controlling Natural Variation in Skin and Flesh Color in Strawberry (Fragaria spp.) Fruit. The Plant cell, 32(12).

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Genotyping and Phylogenetic Analysis of Chlamydia

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Genotyping was performed by amplifying a 990 bp fragment of the ompA gene according to a nested PCR method that was previously described [4 (link)]. The ompA fragments obtained were purified by the FavorPrep™ GEL/PCR Purification Kit (Favorgen Biotech Corporation, Taiwan). In the next step, the products were sent to another laboratory and they were bidirectionally sequenced by the Sanger sequencing method.
In the next step, a BLAST similarity search and a phylogenetic tree analysis were carried out to comprehend the evolutionary relations between clinical strains and reference strains. Each sequence was aligned with an analogous sequence from reference strains. The strains were derived from the GenBank database: GenBank accession numbers: M58938, AF063208, M17343, X62918, X62920, X52557, X52080, AF063199, X16007, AF063200, AF063201, AF063202, AF063203, AF063204, M14738, M36533, and X55700). Chlamydia muridarum MoPn (M64171 was used to form an outgroup [4 (link)]. The phylogenetic tree was illustrated using the maximum-likelihood method in the MEGAX software.

Rajabpour M., Emamie A.D, & Pourmand M.R. (2023). Evaluation of Chlamydia trachomatis Genotypes in Endocervical Specimens by Sequence Analysis of ompA Gene among Women in Tehran. Journal of Tropical Medicine, 2023, 8845565.

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PCR Amplification and Gel Purification

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DNA sequences below 3 kb were amplified via PCR using DreamTaq Green PCR Master Mix (2X) (Thermo Fisher Scientific, Vilnius, Lithuania) or HOT FIREPol GC Master Mix (Solis BioDyne, Tartu, Estonia), otherwise, the high-fidelities Phusion DNA Polymerase or Platinum SuperFi Master Mix (Thermo Fisher Scientific, Vilnius, Lithuania) were used instead. All reactions were set up and performed according to the manufacturer's instructions for a high GC content template. Following PCR, the samples were loaded on a 1% agarose gel and the electrophoresis at 120 V. The fragments sizes were estimated using gene ruler 1 kb Plus DNA Ladder (Thermo Fisher Scientific). Amplicons with the correct size were excised from the gel and purified using Favorprep™ GEL/PCR Purification Kit (Favorgen, Wien, Austria) following the manufacturer's instructions, quantified by Nanodrop (Thermo Fisher, Whaltan, USA), and used for GGA assembly and TOPO subcloning.

Bonturi N., Pinheiro M.J., de Oliveira P.M., Rusadze E., Eichinger T., Liudžiūtė G., De Biaggi J.S., Brauer A., Remm M., Miranda E.A., Ledesma-Amaro R, & Lahtvee P.J. (2022). Development of a dedicated Golden Gate Assembly Platform (RtGGA) for Rhodotorula toruloides. Metabolic Engineering Communications, 15, e00200.

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16S rDNA Amplification and Sequencing

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Universal primers, 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1525R (5′-AAAGGAGGTGATCCAGCC-3′), were used for the PCR amplification of 16S rDNA of PJ85 [41 (link)]. Amplification was conducted in a thermal cycler (Thermo Scientific, Waltham, MA, USA). The PCR reaction conditions were initial denaturation at 95 °C for 5 min followed by 30 cycles of denaturation at 95 °C for 60 s, annealing at 55 °C for 60 s, and extension at 72 °C for 60 s. A final extension was conducted at 72 °C for 7 min. The amplicons were purified from 0.8% agarose gel using a FavorPrep™ GEL/PCR Purification Kit (FAVORGEN, Pingtung City, Taiwan). The purified PCR product was ligated to the terminal transferase activity (TA) cloning vector, and the recombinant plasmid was transformed into Escherichia coli JM109. The recombinant plasmid harboring 16S rDNA was extracted and purified with the FavorPrep™ Plasmid DNA Extraction Kit (FAVORGEN, Taiwan). The purified product was submitted for Sanger sequencing at Macrogen, Korea.

Chanthasena P., Hua Y., Rosyidah A., Pathom-Aree W., Limphirat W, & Nantapong N. (2022). Isolation and Identification of Bioactive Compounds from Streptomyces actinomycinicus PJ85 and Their In Vitro Antimicrobial Activities against Methicillin-Resistant Staphylococcus aureus. Antibiotics, 11(12), 1797.

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Genomic Sequencing of Tilapia Lake Virus

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Total RNA extracted from eight TiLV-infected specimens used for amplification of TiLV genomic sequences in the present study were obtained from our previous works (Dong, Ataguba et al., 2017; (link)Taengphu et al., 2019) (link). Segment 1 of all samples were already sequenced (Taengphu et al., 2019) (link). Thus, the remaining TiLV genome segments 2-10 were individually amplified by RT-PCR, using primers and conditions as previously described (Pulido et al., 2019) (link). After agarose gel electrophoresis, the amplicons were gel purified using FavorPrep GEL/PCR purification kit (Favorgen) before being cloned into pGEM-T easy vector (Promega). Recombinant clones were sequenced using T7 promoter, and SP6 promoter primers (Macrogen, South Korea).

Thawornwattana Y., Dong H.T., Phiwsaiya K., Sangsuriya P., Senapin S., & Aiewsakun P. (2020). Tilapia lake virus (TiLV): Genomic epidemiology and its early origin.

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Nested PCR Amplification and Sequencing of gag Gene

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The DNA samples were amplified a fragment of the gag gene by using nested PCR. The target fragment amplifications were carried out with a final volume of 20 μL containing 0.4 U of DNA polymerase (Phusion™ High–Fidelity DNA Polymerase), 200 μM of each dNTP, and 0.5 μM of each primer. The following PCR conditions were applied: initial denaturation at 98 °C for 1 min, 40 cycles of denaturation at 98 °C for 15 s, annealing at 58 °C for 30 s, and extension at 72 °C for 1 min, and a final extension at 72 °C for 10 min. For the second round, 2 μL of product from the first round was added as a DNA template, and the nested round of PCRs were performed in the same condition. Then, products were visualized on 1.5% agarose gel containing RedSafe™ Nucleic Acid Staining Solution (iNtRON Biotechnology, Gyeonggi-do, Korea). The nested PCR positive samples in agarose gel were purified by using FavorPrep™ GEL/PCR Purification Kit (FAVORGEN Biotech Corporation, Taiwan) and sequenced using the Sanger sequencing method (Sequencer ABI3730XL, Bionics Co., Ltd., South Korea).

Mongkonwattanaporn T., Lertwatcharasarakul P, & Rukkwamsuk T. (2024). Development of in-house ELISA based on recombinant gag proteins of small ruminant lentiviruses isolated from goats in Thailand. Scientific Reports, 14, 3636.

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Amplification and Cloning of MYB10 Promoter

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Gene and promoter amplification were performed using MyFi™ DNA Polymerase (Bioline). FvMYB10-gypsy and Fvb1 FIN12 genomic fragments flanking the deleted region were both amplified with 5Prime PCR Extender System (5Prime) using the provided 10X Tunning Buffer and following the manufacturer instructions.
PCR products <8kb were purified with FavorPrep GEL/ PCR Purification Kit (Favorgen). If >8kb, PCR products were precipitated (30% PEG8000; 30mN MgCl2). Purified products were cloned into the pGEM®-T Easy Vector Systems (Promega) for Sanger sequencing.
Multiple sequence alignment of MYB10 promoter fragments was performed using Geneious algorithm (Gap open penalty=30; Gap extension penalty=0) and used for homology tree construction (Genetic distance model: Jukes-Cantor; Tree build method: Neighbor-Joining), both in the Geneious platform (Kearse et al., 2012) .

Castillejo C., Waurich V., Wagner H., Ramos R., Oiza N., Muñoz P.R., Triviño J.C., Caruana J., Liu Z., Cobo N., Hardigan M.A., Knapp S.J., Vallarino J.G., Osorio S., Martín‐Pizarro C., Posé D., Toivainen T., Hytönen T., Oh Y., Barbey C.R., Whitaker V.M., Lee S., Olbricht K., Sánchez‐Sevilla J.F., & Amaya I. (2020). Allelic Variation ofMYB10is the Major Force Controlling Natural Variation of Skin and Flesh Color in Strawberry (Fragariaspp.) fruit.

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Characterization of FaOMT Promoter Fragments

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For characterization of the FaOMT promoter fragment from selected strawberry accessions, PCR and separation in agarose electrophoresis were performed as previously described for FaOMT-SI/NO marker test. Selected bands of about 500 bp from F. virginiana UC-11 and F. moschata ‘Capron Royale’, and 248 bp from F. × ananassa cv. ‘Aromas’, ‘Candonga’, ‘Elvira’, and ‘Pedrone’ were isolated and purified from the agarose gel using the FavorPrep GEL/PCR purification kit (Favorgen Biotech Corp.) and cloned into the pGEM-T Easy vector (Promega). Five independent clones were sequenced for each of the three accessions and assembled into individual contigs using the SeqMan tool (DNAStar). Sequence analyses and comparisons were carried out using the Lasergen software (DNAStar) and the tool Clustal W2 from EBI-EMBL.

Cruz-Rus E., Sesmero R., Ángel-Pérez J.A., Sánchez-Sevilla J.F., Ulrich D, & Amaya I. (2017). Validation of a PCR test to predict the presence of flavor volatiles mesifurane and γ-decalactone in fruits of cultivated strawberry (Fragaria × ananassa). Molecular Breeding, 37(10), 131.

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