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Lightcycler fast start dna master sybr green mix

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The LightCycler Fast Start DNA Master SYBR Green Mix is a laboratory reagent used for real-time quantitative PCR (qPCR) experiments. It contains all the necessary components for performing sensitive and efficient DNA amplification and detection, utilizing the SYBR Green dye to monitor the amplification process.

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Lab products found in correlation

Random primers, by Cytiva (4 mentions) Mirvana kit, by Thermo Fisher Scientific (2 mentions) Mastercycler realplex2, by Eppendorf (2 mentions) Rt go mastermix, by MP Biomedicals (2 mentions) Trizol reagent, by Thermo Fisher Scientific (1 mentions) Dnase, by Thermo Fisher Scientific (1 mentions) Rneasy mini kit, by Qiagen (1 mentions)

Close spelling variants of this product

LightCycler-Fast Start DNA Master SYBR Green I Mix LightCycler SYBR Green master mix FAST SYBR mix Fast SYBR Green SYBR Master Mix SYBR Green Mix SYBR Green LightCycler

7 protocols using lightcycler fast start dna master sybr green mix

1

qPCR Analysis of CTGF Expression

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RNA was extracted using trizol/chloroform isolation followed by Ambion mirVana kit (AM1560). Total RNA was reverse-transcribed using random primers (Amersham Biosciences), and β-actin primers were used to control for cDNA concentration in a separate PCR reactions for each sample. LightCycler Fast Start DNA Master SYBR Green Mix (Roche) was added to each PCR reaction along with cDNA and 1 pmol primer in a total volume of 10 µl. qPCR was performed on a eppendorf realplex2 mastercycler using the real-time primers provided, according to instructions. Ct values were converted to fold expression changes (2−ΔΔCt values) following normalization to β-actin. Primer sequences are listed below.
GeneSequence
CTGFF 5’-GTGAGTCCTTCCAAAGCAGC-3’
R 5’-TAGTTGGGTCTGGGCCAAAT-3’
β-actinF 5’-GTGGGCCGCCCTAGGCACCA-3’
R 5’-CGGTTGGCCTTAGGGTTCAGGG-3’
Laklai H., Miroshnikova Y.A., Pickup M.W., Collisson E.A., Kim G.E., Barrett A.S., Hill R.C., Lakins J.N., Schlaepfer D.D., Mouw J.K., LeBleu V.S., Roy N., Novitskiy S.V., Johansen J.S., Poli V., Kalluri R., Iacobuzio-Donahue C.A., Wood L.D., Hebrok M., Hansen K., Moses H.L, & Weaver V.M. (2016). Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular-fibrosis and tumor progression. Nature medicine, 22(5), 497-505.
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2

qPCR Analysis of CTGF Expression

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RNA was extracted using trizol/chloroform isolation followed by Ambion mirVana kit (AM1560). Total RNA was reverse-transcribed using random primers (Amersham Biosciences), and β-actin primers were used to control for cDNA concentration in a separate PCR reactions for each sample. LightCycler Fast Start DNA Master SYBR Green Mix (Roche) was added to each PCR reaction along with cDNA and 1 pmol primer in a total volume of 10 µl. qPCR was performed on a eppendorf realplex2 mastercycler using the real-time primers provided, according to instructions. Ct values were converted to fold expression changes (2−ΔΔCt values) following normalization to β-actin. Primer sequences are listed below.
GeneSequence
CTGFF 5’-GTGAGTCCTTCCAAAGCAGC-3’
R 5’-TAGTTGGGTCTGGGCCAAAT-3’
β-actinF 5’-GTGGGCCGCCCTAGGCACCA-3’
R 5’-CGGTTGGCCTTAGGGTTCAGGG-3’
Laklai H., Miroshnikova Y.A., Pickup M.W., Collisson E.A., Kim G.E., Barrett A.S., Hill R.C., Lakins J.N., Schlaepfer D.D., Mouw J.K., LeBleu V.S., Roy N., Novitskiy S.V., Johansen J.S., Poli V., Kalluri R., Iacobuzio-Donahue C.A., Wood L.D., Hebrok M., Hansen K., Moses H.L, & Weaver V.M. (2016). Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular-fibrosis and tumor progression. Nature medicine, 22(5), 497-505.
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3

Quantitative Gene Expression Analysis

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Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed on genes of interest that were up-regulated via microarray analysis. Total RNA was reverse transcribed using random primers (Amersham Biosciences, Little Chalfont, UK), with GAPDH primers used to control for cDNA concentration in separate PCR reactions for each sample. Primers for GADPH, ADAMTS1, LOXL4, BMP2, SMAD7, ITGA2, and TRPV4 were designed using Primer3 and are shown in Supplemental Table 5. To each PCR reaction (triplicates), LightCycler Fast Start DNA Master SYBR Green Mix (Roche, Basel, Switzerland) was added, along with cDNA and 1pmol primer in a total reaction volume of 10μl. Ct values were converted to fold expression changes (2–ΔΔCt values) after normalization to GAPDH expression levels.
Lee J.K., Huwe L.W., Paschos N., Aryaei A., Gegg C.A., Hu J.C, & Athanasiou K.A. (2017). Tension stimulation drives tissue formation in scaffold-free systems. Nature materials, 16(8), 864-873.
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4

Quantitative Gene Expression Analysis

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Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) was performed on genes of interest that were up-regulated via microarray analysis. Total RNA was reverse transcribed using random primers (Amersham Biosciences, Little Chalfont, UK), with GAPDH primers used to control for cDNA concentration in separate PCR reactions for each sample. Primers for GADPH, ADAMTS1, LOXL4, BMP2, SMAD7, ITGA2, and TRPV4 were designed using Primer3 and are shown in Supplemental Table 5. To each PCR reaction (triplicates), LightCycler Fast Start DNA Master SYBR Green Mix (Roche, Basel, Switzerland) was added, along with cDNA and 1pmol primer in a total reaction volume of 10μl. Ct values were converted to fold expression changes (2–ΔΔCt values) after normalization to GAPDH expression levels.
Lee J.K., Huwe L.W., Paschos N., Aryaei A., Gegg C.A., Hu J.C, & Athanasiou K.A. (2017). Tension stimulation drives tissue formation in scaffold-free systems. Nature materials, 16(8), 864-873.
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5

Quantifying DNA Methylation Cleavage

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The extent of cleavage by MSREs was determined using qPCR detection of spiked-in controls. A master mixture (19 μL) containing 2 mM MgCl2, 1× LightCycler FastStart DNA Master SYBR Green mix (Roche Diagnostics Canada, Laval, QC, Canada), 14.4 μL of nuclease-free water and 1 μL of cleaved sample (template) was divided into three fractions to which 0.25 μM of each forward and reverse primer designed to target the appropriate control template was added (see Additional file
2: Table S1). Each qPCR run also included a positive control (non-digested spiked-in, 1/1000 dilution) and negative control (no template). The qPCR conditions were as follows: initial denaturation was carried out at 95°C for 10 minutes, followed by 50 amplification cycles at 95°C for 5 seconds, 52°C for 5 seconds and 72°C for 20 seconds. Melting curve analysis was performed for 1 cycle with a ramp rate of 0.2°C per second, starting at 94°C for 5 seconds, 72°C for 30 seconds, and back to 94°C for 0 seconds, and cooling at 40°C.
Amplicon specificity was determined from the shape of the melting curve and the difference in cycle threshold (Δ Ct) between methylated (MSRE cleavage protected) and unmethylated (MSRE cleavage unprotected) templates was used to calculate cleavage efficiency. The DNA samples were then precipitated by ethanol, washed and dissolved in 10 μL of nuclease-free water.
Shojaei Saadi H.A., O’Doherty A.M., Gagné D., Fournier É., Grant J.R., Sirard M.A, & Robert C. (2014). An integrated platform for bovine DNA methylome analysis suitable for small samples. BMC Genomics, 15(1), 451.
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6

RNA Isolation and RT-qPCR Analysis

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The total RNAs of the cells were isolated using TRIzol reagent (Invitrogen, Carlsbad, California). First-strand synthesis were performed by using RT&Go Mastermix (MP Biomedicals, Aurora, OH) and real-time PCR were performed by a Lightcycler Fast start DNA SYBR Green Master Mix (Roche, Mannheim, Germany) using specific primers with the following sequences (rates normalized to the expression level of actin) : hAPI5, 5;-GGGCAAAAGAGAGCCAGTGA-3′ (forward) and 5′- AAAGTTGCCCAAATTGCTGCT-3′ (reverse); hFGF2, 5′- GGCTATGAAGGAAGATGGAAGATT-3′ (forward) and 5′-TGCCACATACCAACTGGTGTATTT-3′ (reverse); β-Actin, 5′-CGACAGGATGCAGAAGGAGA-3′ (forward) and 5′-TAGAAGCATTTGCGGTGGAC-3′ (reverse).
Noh K.H., Kim S.H., Kim J.H., Song K.H., Lee Y.H., Kang T.H., Han H.D., Sood A.K., Ng J., Kim K., Sonn C.H., Kumar V., Yee C., Lee K.M, & Kim T.W. (2014). API5 confers tumoral immune escape through FGF2-dependent cell survival pathway. Cancer research, 74(13), 3556-3566.
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7

VEGF mRNA Expression Quantification

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Total RNA was extracted with the RNeasy Mini Kit (Qiagen, Gaithersburg, MD) and treated with DNase (Ambion, Austin, TX). cDNA synthesis was performed with RT&Go Mastermix (MP Biomedicals, Aurora, OH), and real-time PCR was performed with Lightcycler FastStart DNA SYBR Green Master Mix (Roche, Mannheim, Germany) using mouse and human Vegfa-specific primers with the following sequences (Bioneer, Daejeon, Korea): mouse Vegfa, 5’-TGCACCCACGACAGAAGGA-3’ (forward) and 5’-GGCAGTAGCTTCGCTGGTAGAC-3’ (reverse); human Vegfa, 5’-CTGCTGTCTTGGGTGCATTGG-3’ (forward) and 5’-CACCGCCTCGGCTTGTCACAT-3’ (reverse). All data was normalized to expression level of β-Actin mRNA.
Lee Y.H., Bae H.C., Noh K.H., Song K.H., Ye S.K., Mao C.P., Lee K.M., Wu T, & Kim T.W. (2015). Gain of HIF-1α under normoxia in cancer mediates immune adaptation through the AKT/ERK and VEGFA axes. Clinical cancer research : an official journal of the American Association for Cancer Research, 21(6), 1438-1446.
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