Quantstudio 6 flex machine

Manufactured by Thermo Fisher Scientific

Sourced in United States

The QuantStudio 6 Flex is a real-time PCR system designed for a wide range of research applications. It features 96-well block format, supports multiple detection chemistries, and provides precise temperature control for reliable results.

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37 protocols using quantstudio 6 flex machine

Quantifying DNA Resection at Double-Strand Breaks

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Cells from each strain were collected from the OCE process and resuspended in 15ml YPLG.
2.5mL of the cells were pelleted as timepoint 0 sample, and 2% GAL was added to the remaining cells, to induce a DSB. Following that, respective timepoint samples were collected.
Genomic DNA was purified using standard genomic preparation method by isopropanol precipitation and ethanol washing, and DNA was re-suspended in 100 mL ddH 2 O. Genomic DNA was treated with 0.005μg/μL RNase A for 45min at 37°C. 2 μL of DNA was added to tubes containing CutSmart buffer with or without the RsaI restriction enzyme and incubated at 37°C for 2hrs. Quantitative PCR was performed using the Applied Biosystem QuantStudio 6 Flex machine. PowerUp SYBR Green Master Mix was used to quantify resection at MAT1 (0.15 kb from DSB) locus. Resection at PRE1 was used as a negative control. RsaI cut DNA was normalized to uncut DNA as previously described to quantify the % ssDNA / total DNA (Ferrari et al. 2015) .

Mojumdar A., Mair N., Adam N., & Cobb J.A. (2022). Changes in DNA double-strand break repair during aging correlate with an increase in genomic mutations.

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Quantifying DNA Resection at DSB

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Cells from each strain were collected from the OCE process and resuspended in 15ml YPLG.
Next, 2.5mL of the cells were pelleted as 'No break' sample, and 2% GAL was added to the remaining cells, to induce a DSB. 2.5ml of cells were pelleted after 3hr incubation as timepoint 0 sample. Following that, GAL was washed off and the cells were released in YPAD and respective timepoint samples were collected. Genomic DNA was purified using standard genomic preparation method by isopropanol precipitation and ethanol washing, and DNA was re-suspended in 100mL ddH 2 O. Quantitative PCR was performed using the Applied Biosystem QuantStudio 6 Flex machine. PowerUp SYBR Green Master Mix was used to quantify resection at HO6 (at DSB) locus. PRE1 was used as a negative control. Signals from the HO6 timepoints were normalized to 'No break' signals and % Ligation was determined.

Mojumdar A., Mair N., Adam N., & Cobb J.A. (2022). Changes in DNA double-strand break repair during aging correlate with an increase in genomic mutations.

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Quantify DNA Resection at DSB Sites

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Cells from each strain were grown overnight in 10ml YPLG to reach an exponentially growing culture of 1×10⁷ cells/mL and were arrest cells in G1 with 15 µg/mL α-factor. 1 hr post α-factor addition to the media a second dose of 15 µg/mL α-factor was added for an additional 1 hr. Next, 2.5 mL of the cells were pelleted as t = 0 and 2% GAL, to induce a DSB, and 15 µg/mL α-factor, to maintain G1 arrest, was added to the remaining cells. 15 µg/mL α-factor was added every 1.5 hr post GAL addition to maintain cells in G1 for the duration of the experiment. 6 hours following GAL addition to the media 2.5 mL of the remaining cells were pelleted as the t = 6 hr time point. Genomic DNA was purified using standard genomic preparation methods and DNA was re-suspended in 100 mL ddH₂O. Genomic DNA was treated with 0.005 µg/µL RNase A for 45 min at 37°C. 2 µL of DNA was added to tubes containing Cut Smart buffer with or without RsaI restriction enzyme and incubated at 37°C for 2 hr. Quantitative PCR was performed using the Applied Biosystem QuantStudio 6 Flex machine. PowerUp SYBR Green Master Mix was used to quantify resection at MAT1 (0.15kb from DSB) and MAT2 (4.8kb from DSB). Pre1 was used as a negative control. RsaI cut DNA was normalized to uncut DNA as previously described to quantify the % ssDNA / total DNA (Ferrari et al., 2015 (link)).

Mojumdar A., Sorenson K., Hohl M., Toulouze M., Lees-Miller S.P., Dubrana K., Petrini J.H, & Cobb J.A. (2019). Nej1 Interacts with Mre11 to Regulate Tethering and Dna2 Binding at DNA Double-Strand Breaks. Cell reports, 28(6), 1564-1573.e3.

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Quantifying DNA Double-Strand Break Resection

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Cells from each strain were grown overnight in 15 ml YPLG to reach an exponentially growing culture of 1 × 10⁷ cells/ml. Next, 2.5 ml of the cells were pelleted as time point 0 sample, and 2% GAL was added to the remaining cells, to induce a DSB. Following that, respective time point samples were collected. Genomic DNA was purified using standard genomic preparation method by isopropanol precipitation and ethanol washing, and DNA was resuspended in 100 ml ddH₂O. Genomic DNA was treated with 0.005 μg/μl RNase A for 45 min at 37 °C. About 2 μl of DNA was added to tubes containing CutSmart buffer with or without inclusion of the RsaI restriction enzyme and incubated at 37 °C for 2 h. qPCR was performed using the Applied Biosystem QuantStudio 6 Flex machine. PowerUp SYBR Green Master Mix was used to quantify resection at the RsaI cut site 0.15 Kb DSB (in the MAT1 locus) and 4.8 Kb. PRE1 was used as a negative control, and the primer sequences are listed in Table S2. RsaI cut DNA was normalized to uncut DNA as previously described to quantify the %ssDNA (28 ). HO cutting was measured in strains for resection (Table S4).

Mojumdar A., Granger C., Lunke M, & Cobb J.A. (2024). Loss of Dna2 fidelity results in decreased Exo1-mediated resection at DNA double-strand breaks. The Journal of Biological Chemistry, 300(3), 105708.

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Quantifying DNA Resection at DSB

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As described previously (9 ), cells from each strain were grown overnight in 15 ml YPLG to reach an exponentially growing culture of 1 × 10⁷ cells/ml. Next, 2.5 ml of the cells were pelleted as “no break” sample, and 2% GAL was added to the remaining cells, to induce a DSB. About 2.5 ml of cells were pelleted after a 3 h incubation as time point 0 sample. After that, GAL was washed off, and the cells were released into YPAD, and respective time point samples were collected. Genomic DNA was purified using standard genomic preparation method by isopropanol precipitation and ethanol washing, and DNA was resuspended in 100 μl ddH₂O. qPCR was performed using the Applied Biosystem QuantStudio 6 Flex machine. PowerUp SYBR Green Master Mix was used to quantify resection at HO6 (at DSB) locus. The PRE1 locus was used as an internal gene control for normalization. Signals from the HO6/PRE1 time points were normalized to “no break” signals, and % Ligation was determined. The primer sequences are listed in Table S2.

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Quantifying DNA Resection Kinetics

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Cells from each strain were grown overnight in 10 ml YPLG to a cell titer of 1x10⁷ cells/ml. 15 μg/mL Nocodazole was added to arrest cells in G2/M. 2 hours post nocodazole addition to the media, 2.5 mL of the cells were pelleted as t = 0 and 2% galactose was added for HO-DSB induction. Other timepoints were taken at t = 1, 2 and 4 hours. Genomic DNA was purified using standard genomic preparation methods and DNA was re-suspended in 100 μl water. Genomic DNA was treated with 5.0 μg/ml RNase A for 45min at 37°C. 2 μl of DNA was added to tubes containing CutSmart buffer with or without RsaI restriction enzyme and incubated at 37°C for 2 hours. Quantitative PCR was performed using the Applied Biosystem QuantStudio 6 Flex machine. PowerUp SYBR Green Master Mix was used to quantify resection at MAT1 (0.15 kb from DSB) and MAT2 (4.8 kb from DSB). Pre1 was used as a negative control. RsaI cut DNA was normalized to uncut DNA as previously described to quantify the percentage of ssDNA per total amount of DNA [52 (link)]. Same primers were used for Q-PCR as previously published [29 (link)].

Hohl M., Mojumdar A., Hailemariam S., Kuryavyi V., Ghisays F., Sorenson K., Chang M., Taylor B.S., Patel D.J., Burgers P.M., Cobb J.A, & Petrini J.H. (2020). Modeling cancer genomic data in yeast reveals selection against ATM function during tumorigenesis. PLoS Genetics, 16(3), e1008422.

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Quantify DNA Resection at DSB Sites

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Quantifying DNA End Resection at DSB

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Cells from each strain were grown overnight in 15 ml YPLG to reach an exponentially growing culture of 1 × 10⁷ cells/ml. Next, 2.5 ml of the cells were pelleted as timepoint 0 sample, and 2% GAL was added to the remaining cells to induce a DSB. Following that, respective timepoint samples were collected. Genomic DNA was purified using standard genomic preparation method by isopropanol precipitation and ethanol washing, and DNA was resuspended in 100 ml ddH₂O. Genomic DNA was treated with 0.005 μg/μl RNase A for 45 min at 37 °C. Two microliters of DNA was added to tubes containing CutSmart buffer with or without RsaI restriction enzyme and incubated at 37 °C for 2 h. Quantitative PCR was performed using the Applied Biosystem QuantStudio 6 Flex machine. PowerUp SYBR Green Master Mix was used to quantify resection at MAT1 (0.15 kb from DSB) locus. Pre1 was used as a negative control and the % resected/cut HO loci is reported from the amount of RsaI cut DNA normalized to the level of HO cutting at each timepoint (Table S1) (36 ).

Mojumdar A., Adam N, & Cobb J.A. (2022). Nej1 interacts with Sae2 at DNA double-stranded breaks to inhibit DNA resection. The Journal of Biological Chemistry, 298(6), 101937.

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Quantifying DNA Resection Dynamics

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Cells from each strain were grown overnight in 15ml YPLG to reach an exponentially growing culture of 1x10 7 cells/mL. Next, 2.5mL of the cells were pelleted as timepoint 0 sample, and 2% GAL was added to the remaining cells, to induce a DSB. Following that, respective timepoint samples were collected. Genomic DNA was purified using standard genomic preparation method by isopropanol precipitation and ethanol washing, and DNA was re-suspended in 100mL ddH2O.
Genomic DNA was treated with 0.005μg/μL RNase A for 45min at 37°C. 2μL of DNA was added to tubes containing CutSmart buffer with or without RsaI restriction enzyme and incubated at 37°C for 2hrs. Quantitative PCR was performed using the Applied Biosystem QuantStudio 6 Flex machine. PowerUp SYBR Green Master Mix was used to quantify resection at MAT1 (0.15kb from DSB) locus. Pre1 was used as a negative control. RsaI cut DNA was normalized to uncut DNA as previously described to quantify the %ssDNA [26] (link). The primer sequences are listed in Table S3.

Mojumdar A., Adam N, & Cobb J.A. (2022). Multifunctional properties of Nej1^XLF C-terminus promote end-joining and impact DNA double-strand break repair pathway choice. DNA repair, 115.

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Quantifying DNA resection dynamics

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Cells from each strain were grown overnight in 15ml YPLG to reach an exponentially growing culture of 1x10 7 cells/mL. Next, 2.5mL of the cells were pelleted as 'No break' sample, and 2% GAL was added to the remaining cells, to induce a DSB. 2.5ml of cells were pelleted after 3hr incubation as timepoint 0 sample. Following that, GAL was washed off and the cells were released in YPAD and respective timepoint samples were collected. Genomic DNA was purified using standard genomic preparation method by isopropanol precipitation and ethanol washing, and DNA was re-suspended in 100uL ddH2O. Quantitative PCR was performed using the Applied Biosystem QuantStudio 6 Flex machine. PowerUp SYBR Green Master Mix was used to quantify resection at HO6 (at DSB) locus. Pre1 was used as an internal gene control for normalization.
Signals from the HO6/Pre1 timepoints were normalized to 'No break' signals and % Ligation was determined. The primer sequences are listed in Table S3.

Mojumdar A., Adam N, & Cobb J.A. (2022). Multifunctional properties of Nej1^XLF C-terminus promote end-joining and impact DNA double-strand break repair pathway choice. DNA repair, 115.

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