qRT-PCR was performed using StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and Kapa SYBR FAST qPCR Master Mix ABI Prism™ (2X, KK4601; Kapa Biosystems, Inc., Wilmington, MA, USA), according to the manufacturer's instructions. SYBR Green assays were performed using 0.5 µl PCR forward primer (10 µM), 0.5 µl PCR reverse primer (10 µM), 2 µl cDNA, 5 µl 2X master mix, and 2 µl double distilled water in a total reaction volume of 10 µl. The PCR protocol was initiated at 95°C for 3 min, followed by 40 cycles of 95°C for 10 sec and 60°C for 60 sec. GAPDH was used as a reference gene. Results were obtained using three independent wells. Relative expression level was calculated using 2−∆∆Ct method (19 (link)) after normalizing with GAPDH expression level. Sequences of primers used for performing qRT-PCR are presented in Table I .
Kapa sybr fast abi prism qpcr master mix
Manufactured by Roche
Sourced in United States
KAPA SYBR FAST ABI Prism qPCR Master Mix is a ready-to-use reaction mix for quantitative real-time PCR (qPCR) on ABI Prism instruments. It contains SYBR Green I dye, a DNA polymerase, dNTPs, and necessary buffer components.
Automatically generated - may contain errors
Lab products found in correlation
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Anti ha, by Santa Cruz Biotechnology (2 mentions)
Abi prism 7900ht sequence detection system, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Transcriptor high fidelity cdna kit, by Roche (1 mentions)
Hotstartaq dna polymerase, by Qiagen (1 mentions)
Thermocycler, by Bio-Rad (1 mentions)
Superscript 3 rt pcr system, by Thermo Fisher Scientific (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Anti h3k4me3, by Active Motif (1 mentions)
Anti h3k36me3, by Active Motif (1 mentions)
Normal mouse igg, by Santa Cruz Biotechnology (1 mentions)
Anti h3, by Abcam (1 mentions)
5 protocols using kapa sybr fast abi prism qpcr master mix
1
SYBR Green qRT-PCR Protocol for Gene Expression
2
RNA Extraction and Quantitative RT-PCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted by RNeasy mini kit (QIAGEN) and treated with DNase I. A half μg of the total RNA was used for cDNA synthesis by Transcriptor High Fidelity cDNA kit (Roche). Quantitative RT-PCR was performed by KAPA SYBR Fast qPCR Master Mix ABI Prism (Kapa Biosystems) by StepOnePlus Real-Time PCR system (Applied Biosystems). The primer sequences are shown in Supplementary Table S3 .
3
ChIP Assay for OsNTL5∆CSRDXOE10 in Rice
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the ChIP assay, 14-day-old seedlings of WT and OsNTL5∆CSRDXOE10 were used as materials. The detailed ChIP experiment was done as described previously (Gan et al., 2014 (link)). Total chromatin was immunoprecipitated using with anti-HA (Santa Cruz Biotechnology, #sc-7392). The mouse IgG (Santa Cruz Biotechnology, #sc-2025) was used as a control ChIP assay. DNA fragments were collected using PCR purification Kit. ChIP-qPCR was performed on the ABI PRISM 7900HT real-time PCR machine (Applied Biosystems) with the KAPA SYBR FAST ABI Prism qPCR Master Mix (KAPA Biosystems). The promoter of UBI (Os01g45400) was used as a negative control. The ChIP relative enrichment of HA-NTL5∆CSRDX10 binding was calculated from the ratio between anti-HA antibody and normal mouse IgG immunoprecipitated DNA (Gan et al., 2014 (link)). The ChIP-qPCR assays were done in triplicate. Primers for the ChIP assays are listed in Supplementary Table 1 .
4
Tissue-specific and Circadian Expression of MRG Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the tissue expression analysis of MRG1 and MRG2, total RNAs were isolated from the root, juvenile rosette leaf, mature rosette leaf, cauline leaf, stem, flower bud, open flower and silique. For the flowering time gene expression analysis, the aerial parts of 6 day-after-germination (DAG) and 9 DAG seedlings were harvested at dusk (ZT16). For a time course assay over a 24-h-long day cycle, the aerial parts of 9 DAG seedlings were harvested every 2 or 4 h. The total RNAs were extracted using an RNeasy plant mini kit (Qiagen) according to the manufacturer's instructions. Approximately 500 ng total RNAs were used for reverse transcription with the Superscript III RT-PCR system (Invitrogen). Semi-quantitative PCR (semi-qPCR) with gene-specific primers (Supplementary Table S1) were performed using HotStarTaq DNA Polymerase (Qiagen) on a Thermocycler (Bio-Rad) at 25–35 cycles. Real-time qPCR was performed on ABI PRISM 7900HT sequence detection system (Applied Biosystems) using KAPA SYBR FAST ABI Prism qPCR Master Mix (KAPA Biosystems). The ubiquitously expressed Tip41-like (AT4G34270) (39 (link)) was used as an internal reference gene. Primer sequences were shown in Supplementary Table S1.
5
ChIP-qPCR analysis of histone marks
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ChIP experiments were performed as previously described with minor modifications (41 (link)). Briefly, total chromatin was extracted from 10 DAG seedlings and immuno-precipitated using anti-HA (Santa Cruz Biotechnology, #sc-7392) and normal mouse IgG as a control (Santa Cruz Biotechnology, #sc-2025), or anti-H3Ace3 (Millipore, #06-599), anti-H4K5Ace (Active Motif #39699), anti-H3K4me3 (Active Motif #61379) and anti-H3K36me3 (Active Motif #61021), with anti-H3 (Abcam, #ab1791) as a control. DNA fragments were recovered by phenol-chloroform extraction and ethanol precipitation. Quantitative PCR with locus-specific primers (Supplementary Table S1) was performed to measure the amounts of FT relative to that of the constitutively expressed ACTIN2 (AT3G18780) on ABI PRISM 7900HT sequence detection system (Applied Biosystems) using KAPA SYBR FAST ABI Prism qPCR Master Mix (KAPA Biosystems).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!