Surface-sterilized seeds of Col-0 and avp2;1 mutants were sown in solid medium containing different concentrations of boric acid for 9 days. Roots were harvested and immediately frozen using liquid nitrogen for RNA extraction. The frozen samples were homogenized at 1700 rpm for 15 sec four times using Multi beads shocker (YASUI KIKAI) in a 3 mL tube. Total RNA was extracted using RNeasy Plant Mini Kit (Qiagen, Germany) and treated with RNase-free DNase (Qiagen, Germany) for 30 mins to eliminate genomic DNA contamination. cDNA was synthesized from 0.25 µg total RNA using the PrimeScript RT reagent kit (TAKARA). Expression level of AVP2;1, NIP5;1, BOR1, and BOR2 was analyzed by qRT-PCR using Thermal Cycle Dice (TAKARA, Japan) with SYBR premix Ex Taq II (TAKARA, Japan). The mRNA level of AVP2;1 was detected at the 5’ (upstream) portion and 3’ (downstream) portion of T-DNA insertion. EF1α was used as a reference gene for normalization of the target mRNA level. Primers used are listed in Table S1
Thermal cycle dice
Manufactured by Takara Bio
Sourced in Japan
The Thermal Cycle Dice is a compact and portable device used for DNA amplification in polymerase chain reaction (PCR) experiments. It features a temperature-controlled block that can accommodate multiple sample tubes or microplates. The Thermal Cycle Dice is designed to precisely control and cycle the temperature of samples, which is a critical step in the PCR process.
Automatically generated - may contain errors
Lab products found in correlation
Primescript rt reagent kit, by Takara Bio (2 mentions)
Sybr premix ex taq 2, by Takara Bio (2 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Rnase free dnase, by Qiagen (1 mentions)
Multi beads shocker, by Yasui Kikai (1 mentions)
Ibright fl1000, by Thermo Fisher Scientific (1 mentions)
Anti c myc a19032, by ABclonal (1 mentions)
C15410174, by Diagenode (1 mentions)
Gtx122148, by GeneTex (1 mentions)
Trizol agent, by Thermo Fisher Scientific (1 mentions)
3 protocols using thermal cycle dice
1
Boric Acid Stress Response Analysis
2
Chromatin Immunoprecipitation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For chromatin immunoprecipitation (ChIP) assays, anti-histone H3, GTX122148 (GENETEX), anti-acetyl histone H3 (H3K27ac), #39134, 39336 (Active Motief), anti-Sp1, GTX110593 (GENETEX), A19649 (ABclonal), anti-c-Myc, A19032 (ABclonal), C15410174 (Diagenode), anti-c-MycK323ac, C15410346 (Diagenode) were used. The detailed procedures were provided in Supplementary Data S1 and Supplementary Table S1 . qPCR analysis was performed using the Thermal Cycle Dice (Takara Bio). Several amplifications were performed by classic PCR and products were run on 1.5% agarose gels and was visualized on iBrightFL1000 (Thermo Fisher Scientific). Primer sequences were available in Supplementary Table S1 .
3
Cardiac Gene Expression Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The messenger (m)RNA levels of CPT-1 and phosphofructokinase (PKF-1) were evaluated by RT-qPCR. Cardiac tissues were homogenized in TRIzol agent (Invitrogen Life Technologies, Carlsbad, CA, USA) and total RNA was extracted and reverse-transcribed into cDNA using the PrimeScript RT reagent kit (Takara Biotechnology, Co., Ltd., Dalian, China) with an Oligo dT primer. All samples were run in triplicate in a total reaction volume of 25 µl, which was comprised of 12.5 µl SYBR Premix Ex TaqⅡ, 2 µl primer, 2 µl templates (1 µg cDNA) and 8.5 µl nuclease-free water. RT-qPCR was performed using the SYBR Premix Ex TaqⅡ (Takara Biotechnology, Co., Ltd.) and two-step Real Time PCR system (Thermal Cycle Dice; Takara Bio, Inc., Otsu, Japan) in triplicate wells. The cycling parameters were as follows: Activation at 95˚C for 30 sec, 40 cycles of denaturation at 95˚C for 5 sec, and then annealing and extension at 60˚C for 30 sec. The rat GAPDH gene was used as a reference. The primers used in the present study are presented in Table I. The normalized fold-changes of the target gene mRNA expression were expressed as 2 -ΔΔCt .
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!