Hotstartaq dna polymerase

Manufactured by Promega

Sourced in United States

HotStarTaq DNA Polymerase is a heat-activated DNA polymerase enzyme used for PCR amplification. It is inactive at lower temperatures and becomes active upon heat activation, which helps to improve specificity and sensitivity of the PCR reaction.

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Lab products found in correlation

Epitect pcr control dna set, by Qiagen (4 mentions) Epitect bisulfite kit, by Qiagen (4 mentions) Dntps, by Promega (4 mentions) Agilent tapestation system, by Agilent Technologies (2 mentions) Hotstartaq master mix, by Qiagen (1 mentions) Mgcl2, by Promega (1 mentions)

5 protocols using hotstartaq dna polymerase

Methylation Analysis of MGMT Promoter

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DNA was extracted using standard phenol/chloroform methods. The purity and concentration of DNA were estimated after collecting absorbance readings at 260/280 nm. DNA (2 μg) was treated with bisulfite (EpiTect Bisulfite Kit, Qiagen, Hilden, Germany). The modified DNA was amplified using primers specific for methylated or unmethylated MGMT gene promoters, as listed in Table S1. Each PCR mixture contained 1 μl of DNA, 500 nM of primers, 1x reaction buffer containing 1.5 mM MgCl₂, and 1 U HotStarTaq DNA Polymerase and 250 mM dNTPs (Promega, USA). PCR was performed with thermal conditions as follows: 95 °C for 10 min, 45 cycles of 95 °C for 30 s, 57 °C for 30 s and 72 °C for 30 s with a final extension of 72 °C for 10 minutes. PCR products were visualized using 1.5% agarose gel, yielding a band of 81 bp for a methylated product and 93 bp for an unmethylated product. Positive methylated and positive unmethylated controls (EpiTect PCR Control DNA Set Qiagen, Hilden, Germany) were included.

Ciechomska I.A., Gielniewski B., Wojtas B., Kaminska B, & Mieczkowski J. (2020). EGFR/FOXO3a/BIM signaling pathway determines chemosensitivity of BMP4-differentiated glioma stem cells to temozolomide. Experimental & Molecular Medicine, 52(8), 1326-1340.

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Rapid PCR Detection of Cronobacter sakazakii

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To amplify the regions of 16S rRNA, rpoB and the seven known C. sakazakii-specific O-antigen genes, nine unique PCR protocols were developed using the published PCR primer sets (Table 3). For 16S rRNA PCR amplification, a total of 50 µL PCR reaction consisted of 25 µL of HotStarTaq Master Mix (QIAGEN, this premixed solution contains HotStarTaq DNA Polymerase, PCR Buffer, and dNTPs with a final concentration of 1.5 mM MgCl₂ and 200 µM each dNTP), and 25 µL of a solution containing 200 nM of each primer, 1.5 mM of additional MgCl₂ (Promega, Madison, WI, USA) and template DNA (50 ng) diluted in PCR grade water. The PCR reactions were run for 35 cycles (each cycle is 94 °C for 45 s, 60 °C for 45 s, and 72 °C for 60 s) in a GeneAmp PCR 9700 thermocycler (Applied Biosystems, Foster City, CA, USA), with an initial hot start (94 °C for 15 min) and a final extension (72 °C for 10 min). The PCR conditions for rpoB and O-antigen amplification were similar to 16S rRNA except that the annealing temperature was 56 °C for rpoB and it was 50 °C for the O-antigen PCR amplification. The PCR products were examined by agarose gel electrophoresis and visualized after ethidium bromide staining (Figure 1).

Miranda N., Banerjee P., Simpson S., Kerdahi K, & Sulaiman I.M. (2017). Molecular Surveillance of Cronobacter spp. Isolated from a Wide Variety of Foods from 44 Different Countries by Sequence Typing of 16S rRNA, rpoB and O-Antigen Genes. Foods, 6(5), 36.

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Quantitative Methylation Analysis of MGMT

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DNA was extracted using standard phenol/chloroform methods. The purity and concentration of DNA were estimated after collecting absorbance readings at 260/280 nm. DNA (2 μg) was treated with bisulfite (EpiTect Bisulfite Kit, Qiagen, Hilden, Germany). The modified DNA was amplified using primers specific for methylated or unmethylated MGMT gene promoters, as listed in Additional file 1: Table S4. Each PCR mixture contained 1 μl of DNA, 500 nM of primers, 1 × reaction buffer containing 1.5 mM MgCl2, and 1 U HotStarTaq DNA Polymerase and 250 mM dNTPs (Promega, USA). PCR was performed with thermal conditions as follows: 95 °C for 10 min, 45 cycles of 95 °C for 30 s, 57 °C for 30 s and 72 °C for 30 s with a final extension of 72 °C for 10 min. PCR products were visualized using Agilent Tape Station system (Agilent Technologies, Palo Alto, CA, USA), yielding a band of 81 bp for a methylated product and 93 bp for an unmethylated product. Positive methylated and positive unmethylated controls (EpiTect PCR Control DNA Set Qiagen, Hilden, Germany) were included.

Roura A.J., Szadkowska P., Poleszak K., Dabrowski M.J., Ellert-Miklaszewska A., Wojnicki K., Ciechomska I.A., Stepniak K., Kaminska B, & Wojtas B. (2023). Regulatory networks driving expression of genes critical for glioblastoma are controlled by the transcription factor c-Jun and the pre-existing epigenetic modifications. Clinical Epigenetics, 15, 29.

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Determination of MGMT Gene Promoter Methylation

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DNA was extracted using standard phenol/chloroform methods. The purity and concentration of DNA was estimated at the absorbance of 260 and 280 nm. DNA (2 μg) was treated with chemical bisulfite modification (EpiTect Bisulfite Kit, Qiagen, Hilden, Germany) to convert unmethylated, but not the methylated, cytosines to uracil. The modified DNA was then amplified using primers specific for either methylated or unmethylated MGMT gene promoter sequences, as listed in Supplementary Table S1. Each PCR mixture contained 1 μl of DNA, 500 nM of primers, 1x reaction buffer containing 1.5 mM MgCl_2, 1 U HotStarTaq DNA Polymerase and 250 mM dNTPs (Promega, United States). PCR was performed with thermal conditions as: 95°C for 10 min, 45 cycles of 95°C for 30 s, 57°C for 30 s and 72°C for 30 with a final extension of 72°C for 10 min. The PCR reaction was carried out in two separate tubes specific for methylated and unmethylated sequences. The PCR products were visualized using 1.5% agarose gel or Agilent Bioanalyzer yielding a band of 81 bp for a methylated and 93 bp for an unmethylated product. Positive methylated and positive unmethylated controls (EpiTect PCR Control DNA Set Qiagen, Hilden, Germany) are used to confirm methylation evaluation assays.

Ciechomska I.A., Marciniak M.P., Jackl J, & Kaminska B. (2018). Pre-treatment or Post-treatment of Human Glioma Cells With BIX01294, the Inhibitor of Histone Methyltransferase G9a, Sensitizes Cells to Temozolomide. Frontiers in Pharmacology, 9, 1271.

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Quantification of MGMT Promoter Methylation

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DNA was extracted using standard phenol/chloroform methods. The purity and concentration of the DNA were estimated by measuring absorbance at 260/280 nm. DNA (2 μg) was treated with bisulfite (EpiTect Bisulfite Kit, Qiagen, Hilden, Germany). The modified DNA was amplified using primers specific for the methylated or unmethylated MGMT gene promoter, as listed in Supplementary Table S1. Each PCR mixture contained 1 μL of DNA, 500 nM of primers, 1 reaction buffer containing 1.5 mM MgCl₂, and 1 U HotStarTaq DNA polymerase and 250 mM dNTPs (Promega, Madison, WI, USA). PCR was performed with thermal conditions as follows: 95 °C for 10 min, 45 cycles of 95 °C for 30 s, 57 °C for 30 s and 72 °C for 30 s, with a final extension of 72 °C for 10 min. PCR products were visualized using Agilent TapeStation system (Agilent Technologies, Santa Clara, CA, USA) yielding a band of 81 bp for a methylated product and 93 bp for an unmethylated product. Positive methylated and positive unmethylated controls (EpiTect PCR Control DNA Set Qiagen, Germany) were included.

Ciechomska I.A., Wojnicki K., Wojtas B., Szadkowska P., Poleszak K., Kaza B., Jaskula K., Dawidczyk W., Czepko R., Banach M., Czapski B., Nauman P., Kotulska K., Grajkowska W., Roszkowski M., Czernicki T., Marchel A, & Kaminska B. (2023). Exploring Novel Therapeutic Opportunities for Glioblastoma Using Patient-Derived Cell Cultures. Cancers, 15(5), 1562.

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