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Primer mix

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Primer mix is a pre-formulated solution containing the necessary components for DNA amplification reactions, such as oligonucleotide primers, dNTPs, and buffer. It is designed to facilitate efficient and consistent PCR (Polymerase Chain Reaction) or qPCR (quantitative PCR) experiments.

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Lab products found in correlation

Methyledge bisulfite conversion system, by Promega (1 mentions) Amplitaq gold dna polymerase, by Thermo Fisher Scientific (1 mentions) Hot firepol blend master mix ready to load, by Solis BioDyne (1 mentions) Xpert carba r kit, by Cepheid (1 mentions) Dntp mix, by Promega (1 mentions) Whatman, by Cytiva (1 mentions) Gel imaging system, by Syngene (1 mentions) Xp thermal cycler, by Bioer (1 mentions)

4 protocols using primer mix

1

Bisulfite Conversion and PCR Amplification

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Bisulfite conversion of 100 ng of extracted genomic DNA was carried out with the MethylEdge™ Bisulfite Conversion System (Promega) following manufacturer's guidelines, obtaining an elution volume of 20 µL. A PCR multiplex amplification in 10.7 µL reaction volume adding 1.5 µL of converted DNA was carried out using 0.3 µL of 250 U AmpliTaq Gold™ DNA Polymerase, 1.5 µL of 10X Buffer II, 3.9 µL of 25 mM MgCl 2 (all from Applied Biosystems, AB), 1.5 µL of 32 ng/µL bovine serum albumin, 1 µL of 10 mM GeneAmp® dNTP Mix with dTTP (AB) and 1 µL of primer mix (0.083-5 µM of each primer, Metabion International). PCR cycling used a GeneAmp® PCR system 2720 (AB) with cycling conditions: 95ºC for 11 min; 34 cycles of 94ºC for 20 s, 56ºC for 60 s and 72ºC for 30 s, and a final extension of 72ºC for 7 min After checking amplification yields in 1 % agarose gels, a purification of 2.5 µL of PCR product was performed adding 1 µL of ExoSAP-IT™ PCR Product Cleanup Reagent (AB) at 37 °C for 45 min and 80 °C for 15 min
Ambroa-Conde A., Girón-Santamaría L., Mosquera-Miguel A., Phillips C., Casares de Cal M.A., Gómez-Tato A., Álvarez-Dios J., de la Puente M., Ruiz-Ramírez J., Lareu M.V, & Freire-Aradas A. (2022). Epigenetic age estimation in saliva and in buccal cells. Forensic science international. Genetics, 61.
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2

Molecular Detection of Carbapenemase Genes

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On the GeneXpert device, IMP1, VIM, NDM, KPC, and OXA-48 genes were studied with the Xpert Carba-R kit (Cepheid Inc., USA) as per the guidelines of the manufacturer. Identified resistance genes were verified with the multiplex PCR and gel imaging method. Additionally presence of NDM-1 genes in the strains were verified by multiplex-PCR (Fig. 1).

Results of multiplex PCR-gel imaging on K. pneumoniae strains

The five most common carbapenemase genes (blaOXA-48, blaNDM-1, blaIMP, blaVIM, and blaKPC) were investigated by an in-house multiplex PCR test. We used the primers which were previously described in the study by Poirel et al. [12 (link)]. The DNA was extracted by boiling the strains at 95 °C for 5 min. The PCR mixture was constituted of 4 μL master mix (5× HOT FIREPol Blend Master Mix Ready to Load, Solis BioDyne, Estonia), 12 μL dH2O, 1 μL primer mix (metabion GmBh, Germany), and 3 μL bacterial DNA (total volume 20 μL). The PCR protocol followed was 10 min at 94 °C denaturation; 35 cycles of 30 s at 94 °C, 40 s at 56 °C, and 50 s at 72 °C; and final extension for 5 min at 72 °C. PCR products were dyed with SYBR gold, loaded into 2 % agarose gel, and run on an observable real time electrophoresis (ORTE) instrument (Salubris Technica, Istanbul).
Karabay O., Altindis M., Koroglu M., Karatuna O., Aydemir Ö.A, & Erdem A.F. (2016). The carbapenem-resistant Enterobacteriaceae threat is growing: NDM-1 epidemic at a training hospital in Turkey. Annals of Clinical Microbiology and Antimicrobials, 15, 6.
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3

Multiplex Mitochondrial DNA Barcoding

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For this method, primers complementary to mitochondrial DNA cytochrome b were used [12 (link),24 ]. mPCR amplification was conducted in 15 µL 1.5-mM MgCl2, 0.2-mM dNTP mix (Promega, Madison, WI, USA), primer mix (Metabion International AG, Planegg, Germany), 100 ng template DNA and 0.4 U PlatinumTM DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA). The primers were mixed in the ratio of 1:0.6:0.6:1.5:1.5 for SIM:B:P:C:H and used together to mPCR (ratio 1 means concentration 0.4 μmol∙L−1). Amplifications were performed in a Biometra T-Gradient (Whatman Biometra, Göttingen, Germany) as follows: initial denaturation at 94 °C for 2 min, 40 cycles of denaturation at 94 °C for 30 s, annealing at 53 °C for 30 s and extension at 72 °C for 30 s, final polymerization was for 5 min at 72 °C. Visualization and detection of amplicons were done on 2.5% agarose gel.
Hrbek V., Zdenkova K., Jilkova D., Cermakova E., Jiru M., Demnerova K., Pulkrabova J, & Hajslova J. (2020). Authentication of Meat and Meat Products Using Triacylglycerols Profiling and by DNA Analysis. Foods, 9(9), 1269.
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4

Genetic Profiling of OXPHOS Pathways

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The SNP regions examined in this study are located in genes that play important roles in the OXHPOS pathway and are classified as "benign and likely benign" according to recent guidelines [11] (Tab.1).
In this research, one 0.5 ml PCR tube was labeled for each individual. 50 µl of the mixture was prepared for PCR. 5 µl DNA solution, 10 µl sterile double distilled water, 14 µl PCR master mix and 1.5 µl primer mix (Metabion, Germany) were used for the area concerned. While preparing the primary mixture; 5 µl of each forward and reverse primer of the relevant region was taken and diluted with 90 µl distilled water (Tab.2). In this study, XP Thermal Cycler (Bioer, China) device was used. PCR was performed with 35 cycles of denaturation: at 95 ° C for 1 min; annealing: at 58 ° C for 1 min; elongation: at 72 ° C for 1 min". From obtained products, 4 µl of each was taken and mixed with 0.4 µl DNA dye (Metabion, Germany) and it was run at 120 V current for 20 minutes using 1.8% agarose gel in an electrophoresis system (Wealtec, USA). At the gel-imaging system (Syngene, USA) the band lengths were compared with marker DNA (Thermo Fermentas, Turkey) under UV light. It was decided that the PCR process was successful for the samples with the desired band lengths.
Düzkale N., Kahraman Ç.Y., KİKİ İ., Yıldırım R., Sincan G., & Tatar A. (2021). Investigation of mitochondrial DNA polymorphisms in patients with hematological malignancy. Turkish Journal of Clinics and Laboratory, 12(2), 147-154.
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