Pcr buffer

Manufactured by Merck Group

Sourced in United States, Germany

PCR buffer is a solution used in polymerase chain reaction (PCR) processes to maintain the optimal pH and ionic conditions for the enzymatic amplification of DNA sequences. It provides a suitable environment for the DNA polymerase enzyme to function effectively during the PCR reaction.

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Lab products found in correlation

Mgcl2, by Merck Group (8 mentions) Taq dna polymerase, by Merck Group (5 mentions) Taq polymerase, by Merck Group (4 mentions) Jumpstart taq dna polymerase, by Merck Group (3 mentions) Dntps, by Thermo Fisher Scientific (3 mentions) Dntps, by Merck Group (2 mentions) Redtaq dna polymerase, by Merck Group (1 mentions) Organic solvents, by Merck Group (1 mentions) Yeast extract, by Liofilchem (1 mentions) Tryptone, by Liofilchem (1 mentions) Las 3000 cooled ccd camera, by Fujifilm (1 mentions) Genomelab gexp genetic analysis system, by Beckman Coulter (1 mentions) Chemidoc system, by Bio-Rad (1 mentions) Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Ptc 200 thermal cycler, by Bio-Rad (1 mentions) Magnesium chloride, by Merck Group (1 mentions) Sybr green, by Thermo Fisher Scientific (1 mentions) Cdna synthesis kit, by Thermo Fisher Scientific (1 mentions) Plasmid extraction kit, by Qiagen (1 mentions)

Close spelling variants of this product

LongAmp PCR buffer (2 citations)

21 protocols using pcr buffer

Nested PCR for Toxoplasma Detection

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Nested, conventional PCR targeting the B1 gene (figure 1B) was performed at the Francis I. Proctor Foundation CLIA-accredited microbiology laboratory as part of routine clinical testing. Briefly, intraocular fluid samples were boiled for 10 min and 4 µL of the boiled aqueous or vitreous fluid was subjected to 2 rounds of PCR with the following primers targeting the B1 gene: 5F_external: GGA ACT GCA TCC GTT CAT GAG; 5R_external: TCT TTA AAG CGT TCG TGG TC; 5F_internal: TGC ATA GGT TGC AGT CAC TG; 5R_internal: GGC GAC CAA TCT GCG AAT ACA CC. Both PCR assays used 5 µL of 10X PCR buffer (Sigma-Aldrich, St. Louis, Missouri, USA), 1.5 mM of MgCl₂, 0.1 mM dNTP mix, 10 pmol of each primer and 0.05 U of REDTaq DNA polymerase (Sigma-Aldrich) in a total reaction volume of 50 µL. Amplified products were evaluated on 4% e-Gels (Thermo Fisher Scientific, Waltham, Massachusetts, USA). This assay has a LLOD of 5 tachyzoites/mL (unpublished, F. I. Proctor Foundation).
Samples in which the nested PCR and dual-target, real-time PCR results were discrepant, were tested by B1 real-time PCR at the Palo Alto Medical Foundation Toxoplasmosis Serology Laboratory (PAMF-TSL), the US National Toxoplasmosis Reference Laboratory.

Gomez C.A., Sahoo M.K., Kahn G.Y., Zhong L., Montoya J.G., Pinsky B.A, & Doan T. (2019). Dual-target, real-time PCR for the diagnosis of intraocular Toxoplasma gondii infections. The British Journal of Ophthalmology, 103(4), 569-572.

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Molecular Diagnostics Protocol Optimization

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Tris, tryptone, yeast extract and casein were purchased from Liofilchem (Italy). Materials required for PCR such as 10X PCR buffer, dNTP (10mM), MgCl 2 (20 mM) and Taq DNA polymerase were purchased from Sigma-Aldrich and other required chemicals from Merck (Germany). The required primers were also synthesized by Bioneer (Korea). The organic solvents were purchased from Merck.

Liao X., Teng L, & Li W. (2021). Isolation and production of organic solvent tolerant protease from bacterial burn infection, Staphylococcus aureus KP091274. Cellular and molecular biology (Noisy-le-Grand, France), 67(2).

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Genotyping Atg7 Knockout Mice Using Cre and Flox Markers

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Floxed Atg7 mice were characterized previously¹⁵ (link) and were crossed with a Nes-Cre-driven line to produce Atg7^{flox/flox; Nes-Cre} knockout (atg7 KO) and Atg7^{flox/+; Nes-Cre} control mice (Ctrl). Genomic DNA was isolated from tail samples according to the manufacturer's instruction (Qiagen, 69506). The following primer sets were used for genotyping: for the Cre transgene (Cre-S:cre sense 55) 5′-TTT GCC TGC ATT ACC GGT CGA TGC AAC-3′ and (Cre-As:Cre AS1000) 5′-TGC CCC TGT TTC ACT ATC CAG GTT ACG GA-3′ and for the Ctrl and Atg7^flox alleles (Hind-Fw) 5′-TGG CTG CTA CTT CTG CAA TGA TGT-3′ and (Pst-Rv) 5′-CAG GAC AGA GAC CAT CAG CTC CAC-3′. The reaction mixture for genotyping contained 1 μL of genomic DNA, 0.2 mM dNTP, 2.5 μL 10× PCR buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mM MgCl₂, all from Sigma), 1 U of Taq DNA Polymerase (Sigma, D1806), and 0.5 μM of primer. The PCR parameters were 98°C for 20 s, 64°C for 30 s, and 72°C for 90 s for 30 cycles. PCR products were separated on a 1.5% agarose gel containing SYBR Green. A 100-base pair (bp) ladder was used to verify the size of the PCR products. The gels were imaged with an LAS 3000 cooled CCD camera (Fujifilm, Tokyo, Japan). atg7 KO mice were identified by the presence of both 500 bp and 1000 bp DNA bands. Ctrl mice were identified by the presence of 500 bp, 1000 bp, and 1500 bp products.

Xie C., Ginet V., Sun Y., Koike M., Zhou K., Li T., Li H., Li Q., Wang X., Uchiyama Y., Truttmann A.C., Kroemer G., Puyal J., Blomgren K, & Zhu C. (2016). Neuroprotection by selective neuronal deletion of Atg7 in neonatal brain injury. Autophagy, 12(2), 410-423.

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Multiplex Gene Expression Analysis by GeXP PCR

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The GeXP PCR reaction system contained a total volume of 25 μL, including 2.5 μL of 10 × PCR Buffer (Sigma, STL, MO, USA), 2.5 μL of MgCl₂ (25 μM, Sigma), 1.25 μL of universal primers (500 nmol/L), 1.25 μL of JumpStart Taq DNA polymerase (2.5 U/μL, Sigma), 1.25 μL of mixed primers (containing 50–150 nmol/L of nine gene-specific chimeric primer pairs), and 0.5 pg-0.5 ng of template (cDNA or DNA). Next, nuclease-free water was used to bring the final volume of the PCR reaction to 25 μL. The PCR thermocycling procedure was performed at 95 °C for 5 min, followed by 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s (10 cycles). The second step was performed at 95 °C for 30 s, 65 °C for 30 s, and 72 °C for 30 s (10 cycles). The third step was performed at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s (20 cycles). Detailed information regarding the primers is listed in Table 1; all primers were synthesised and purified by Invitrogen (Guangzhou, China).
The GenomeLab GeXP genetic analysis system (Beckman Coulter, Brea, CA, USA) was used to separate and detect the PCR products by capillary electrophoresis following previous protocols^{43 (link)}. After separating the products, the product peaks were analysed using the GeXP system software. The peak height for each gene is illustrated in an electrophoretogram.

Li M., Xie Z., Xie Z., Liu J., Xie L., Deng X., Luo S., Fan Q., Huang L., Huang J., Zhang Y., Zeng T, & Wang S. (2018). Simultaneous detection of eight avian influenza A virus subtypes by multiplex reverse transcription-PCR using a GeXP analyser. Scientific Reports, 8, 6183.

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Genotyping WT and KO Mice by PCR

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Genotyping was done by polymerase chain reaction (PCR) to distinguish between WT and KO mice. DNA was extracted using Extract N Amp Tissue extraction kit. PCR was set up using 1X PCR buffer from Sigma using the primers 5ʹ-GGATTTGACTTAATTCCTTCAGCGG-3ʹ (wild type reverse), 5ʹ-TCTTGGGCTCGATCTTGTGTCAGGAACAGG-3ʹ (common) and 5ʹ-TGGATGTGGAATGTGTGCGAG-3ʹ (mutant reverse). The PCR sequences were obtained from Jackson Laboratory genotyping database/repository.

Yakkundi P., Gonsalves E., Galou-Lameyer M., Selby M.J, & Chan W.K. (2019). Aryl hydrocarbon receptor acts as a tumor suppressor in a syngeneic MC38 colon carcinoma tumor model. Hypoxia, 7, 1-16.

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Genotyping of NQO1 and SIRT1 Knockout Mice

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Genotyping was done by PCR to distinguish between WT and KO mice (

Supplementary Figure S1

). DNA was extracted using Extract N AMP Tissue extraction kit. PCR was set up using 1X PCR buffer from Sigma using the following primers: 5’-TGTGTACCGTGTGTATGCAA-3’ (NQO1-WT forward), 5’-CTAAGACCTGGAAGCCACAG-3’ (NQO1-WT reverse) resulting 450 bps, 5’-GAAGGGACTGGCTGCTATTG-3’ (NQO1-KO forward), 5’-AATTCACGGGTAGCCAACG-3’ (NQO1-KO reverse) resulting 400 bps, 5’-CATCTAAACTTTGTTGGCTGC-3’ (SIRT1-wild type reverse), 5’-TCCTTGCCACAGTCACTCAC-3’ (SIRT1-common forward), 5’-ACAGTCCCATTC CCATACC-3’ (SIRT1-floxed reverse) resulting 600 bps for WT and 700 bps for KO. Electrophoresis was conducted on 2% agarose gels and visualized with a Bio-Rad ChemiDoc system (Bio-Rad, USA).

Lee S.H., Kim H.J., Oh G.S., Lee S.B., Khadka D., Cao W., Choe S.K., Shim H., Kim C.D., Kwak T.H, & So H.S. (2022). Augmentation of NAD+ by Dunnione Ameliorates Imiquimod-Induced Psoriasis-Like Dermatitis in Mice. Journal of Inflammation Research, 15, 4623-4636.

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Quantitative RT-PCR Analysis of CAIX

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Total RNA was isolated from cells using the RNeasy Mini kit (Qiagen, Hilden, Germany) and the quantity of total RNA was measured using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). cDNA synthesis was performed using oligo (dT) 15 primer and GoScript Transcriptase according to manufacturer’s instructions (Promega GmbH, Mannheim, Germany). The PCR mixture contained: 1.5 uL of cDNA, 1× PCR buffer (Sigma Aldrich) 2.5 mM Magnesium Chloride (Sigma Aldrich), 0.2 mM dNTPs (Sigma Aldrich), 0.2 uM of each primer (Sigma Aldrich), and 1.25 U JumpStart™ Taq DNA Polymerase (Sigma Aldrich). The cDNA was amplified by PCR using the MJ Research PTC-200 Thermal Cycler with the following primers: CAIX (Carbonic Anhydrase 9) forward (5′-TACAGCTGAACTTCCGAGCG-3′), CAIX reverse (5′-CTAGGCTCCAGTCTCGGCTA-3′), HPRT1 (Hypoxanthine-Guanine Phosphoribosyltransferase) forward (5′-TGGCGTCGTGATTAGTGATG-3′), HPRT1 reverse (5′-TATCCAACACTTCGTGGGGT-3′). The PCR conditions for all analyzed genes were as following: denaturing at 95 °C for 5 min, followed by 30 cycles of 30 s at 95 °C, 30 s at 58 °C and 30 s at 72 °C. The reaction was completed for 10 min at 72 °C. The PCR reaction was evaluated by checking the PCR products on 1.5% w/v agarose gels. Bands were normalized by use of HPRT1 to correct for differences in loading of the cDNAs.

Tyszka-Czochara M., Lasota M, & Majka M. (2018). Caffeic Acid and Metformin Inhibit Invasive Phenotype Induced by TGF-β1 in C-4I and HTB-35/SiHa Human Cervical Squamous Carcinoma Cells by Acting on Different Molecular Targets. International Journal of Molecular Sciences, 19(1), 266.

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Detailed Biochemical Protocols for Cellular Studies

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For PCR-based work, materials like PCR buffer and Taq polymerase were purchased from Sigma Aldrich, USA, and dNTPs were from Fermentas, USA. For expression studies, materials like cDNA synthesis kit, SYBR green, and ROX solution were purchased from Fermentas, USA. RNA later was purchased from Sigma Aldrich, USA. For cell culture studies, DMEM, FBS, and metformin were purchased from Sigma Aldrich, USA. LB agar and LB broth were purchased from Himedia; DPnI from Thermo scientific; A769962 from Abcam; Q5 polymerase from New England Biolabs and Plasmid extraction kit from Qiagen. For western blot analysis, phospho-AMPK rabbit monoclonal antibody, total AMPK rabbit monoclonal antibody, and beta-actin rabbit monoclonal antibody were purchased from CST, USA. The anti-rabbit IR DYE 800 antibody was purchased from Li-COR Biosciences, USA.

Kawoosa F., Shah Z.A., Masoodi S.R., Amin A., Rasool R., Fazili K.M., Dar A.H., Lone A, & ul Bashir S. (2022). Role of human organic cation transporter-1 (OCT-1/SLC22A1) in modulating the response to metformin in patients with type 2 diabetes. BMC Endocrine Disorders, 22, 140.

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Detect mtDNA m.5789T>C Variant

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The m.5789T>C variant was detected by amplifying a 186-bp PCR product using primers 5694F (5′-AACAGCTAAGCACCCTAATCAACT-3') and 5879R (5′-GAGTGAAGCATTGGACTGTAAATCT-3′). The PCR mix contained 10 ng of DNA, 0.2 U JumpStart Taq DNA polymerase, 0.1 mM of each dNTP, 1× PCR buffer (Sigma-Aldrich, St. Louis), 2.5 mM MgCl₂, and 250 nM of each primer in a total volume of 25 µL. Amplification conditions were 95°C for 10 minutes; 35 cycles of 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 40 seconds; and finally 72°C for 7 minutes. Amplification products were digested with the restriction enzymes Taq^αI or Hpy188I (New England Biolabs, Ipswich, MA) using the producer's protocol. Taq^αI cuts the wild-type molecules into 2 fragments of 89 bp and 97 bp length, whereas it does not cut the mtDNA molecules carrying the m.5789T>C variant. Hyp188I cuts the wild-type mtDNA into 2 fragments of 58 bp and 128 bp length and the mutated mtDNA into 3 fragments of 58 bp, 31 bp, and 97 bp length. Restriction digestion fragments were separated on a 10% polyacrylamide gel and visualized by SYBR Green I staining (Sigma-Aldrich) and fluorescence detection using a GelDoc system (Bio-Rad Laboratories, Hercules). Proportions of wild-type and mutant mtDNA were estimated from band intensities using ImageJ software. Heteroplasmy values from Taq^αI and Hpy188I restriction digestions were averaged.

Hippen M., Zsurka G., Peeva V., Machts J., Schwiecker K., Debska-Vielhaber G., Wiesner R.J., Vielhaber S, & Kunz W.S. (2021). Novel Pathogenic Sequence Variation m.5789T>C Causes NARP Syndrome and Promotes Formation of Deletions of the Mitochondrial Genome. Neurology: Genetics, 8(2), e660.

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ACE Genotyping by PCR Amplification

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PCR amplification of the polymorphic region of the ACE gene containing either the insertion (I) or dele-tion (D) fragment was performed. Only one pair of primers (ACEfor: CTG GAG ACC ACT CCC ATC CTT TCT and ACErev: GAT GTG GCC ATC ACA TTC GTC AGA) was used to determine the ACE genotype, yielding amplification products of approximately 490 bp (for I allele) and 190 bp (for D allele). The 10 μl PCR consisted of: 1 μl DNA isolate; 0.5 U DNA recombinant Taq polymerase in buffer (pH = 8.0; Sigma, Germany); 1x PCR buffer (pH = 8.7; Sigma, Germany); 1.5 mM MgCl 2; 4 pM primer ACEfor and ACErev (Oligo, Poland) in TE buffer (pH = 8); 0.75 nM of each dNTP. The thermal-time PCR was as follows: initial denaturation at 94^°C for 300 s, 30 cycles (denaturation at 92^°C for 60 s, primer annealing at 58^°C for 60 s, chain extension at 72^°C for 150 s) and final extension at 72^°C for 360 s. The reaction was performed in two samples per isolate. Amplification products were visualized in UV light by using 1.5% agarose gels stained with ethidium bromide.

Grenda A., Leońska-Duniec A., Kaczmarczyk M., Ficek K., Król P., Cięszczyk P, & Żmijewski P. (2014). Interaction Between ACE I/D and ACTN3 R557X Polymorphisms in Polish Competitive Swimmers. Journal of Human Kinetics, 42, 127-136.

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