Genomic DNA editing was detected using a Surveyor Mutation Detection Kit for Standard Gel Electrophoresis (Integrated DNA Technologies) according to the manufacturer's instructions. Briefly, genomic DNA was purified, followed by PCR, and PCR fragments were extracted. DNA was annealed in 1× prime star buffer (Takara) and the surveyor enzyme was added to digest the annealed DNA fragments, followed with gel electrophoresis to detect the DNA bands after digestion.
Primestar buffer
Manufactured by Takara Bio
Sourced in Japan
PrimeSTAR buffer is a specialized buffer solution developed by Takara Bio for use in high-fidelity DNA amplification. It is designed to optimize the performance of the PrimeSTAR HS DNA Polymerase, which is known for its accuracy and efficiency in PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Primestar hs dna polymerase, by Takara Bio (3 mentions)
Surveyor mutation detection kit for standard gel electrophoresis, by Integrated DNA Technologies (2 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Sephadex g 50 column, by GE Healthcare (1 mentions)
Abi 3500 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
Geneamp pcr system 9700 thermal cycler, by Thermo Fisher Scientific (1 mentions)
Bigdye terminator cycle sequence kit v 3, by Thermo Fisher Scientific (1 mentions)
Dneasy plant mini kit, by Qiagen (1 mentions)
C1000 touch thermal cycler, by Bio-Rad (1 mentions)
Emerald luc, by Toyobo (1 mentions)
Dpni, by Takara Bio (1 mentions)
6 protocols using primestar buffer
1
Surveyor Assay for Genome Editing
2
Detecting Genomic DNA Editing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA editing was detected using Surveyor® Mutation Detection Kit for Standard Gel Electrophoresis (Integrated DNA Technologies) according to manufacturer’s instructions. Briefly, genomic DNA was extracted by DNeasy Blood & Tissue Kit (Qiagen). PCR fragments were purified by GeneJET PCR Purification Kit (K0701). After that, PCR fragments were annealing in 1× prime star buffer (Takara) and Surveyor enzyme was added to digest the annealed DNA fragments at 42 °C for 1 h. Gel Electrophoresis on agarose gel was used to detect the digestion.
3
Sequencing of cpDNA spacer regions in Erianthus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Three non-coding intergenic spacer regions of cpDNA—rps16–trnQ, atpA–rps14, and rpl16–rps3—were sequenced in all Erianthus accessions by using Sanger sequencing of each PCR product. Sequence variations in these regions were identified between E. arundinaceus accessions from Japan and Indonesia [52 (link)]. Primer pairs to amplify each region (Table S5 ) were designed from the chloroplast genome sequence of ‘JW630’ (GenBank accession No. LC160130). PCR was performed in a 15-μL mixture containing genomic DNA (20 ng), 5× PrimeSTAR buffer (TaKaRa, Shiga, Japan), 0.4 mM each dNTP (TaKaRa), 5 pmol of each specific forward and reverse primer, and 0.5 units PrimeSTAR HS-DNA polymerase (TaKaRa) in a GeneAmp PCR System 9700 thermal cycler (Life Technologies) as follows: 98 °C for 1 min, followed by 30 cycles of 98 °C for 15 s, 56 °C for 15 s, and 72 °C for 2.5 min. Amplification products were purified with a QuickStep2 PCR Purification Kit (Edge Biosystems, Gaithersburg, MD, USA) and were used as templates for sequencing. Cycle-sequencing was performed with a BigDye Terminator Cycle Sequence Kit v. 3.1 (Life Technologies) using specific primers (Table S5 ) in the same thermal cycler. Sequencing products were purified on a Sephadex G-50 column (GE Healthcare, Uppsala, Sweden) and sequenced in an ABI3500 genetic analyzer (Life Technologies).
4
Detecting CmDFR gene deletion in chrysanthemum
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Genomic DNA was extracted from the leaves of the 22 chrysanthemum cultivars using a DNeasy Plant Mini Kit (Qiagen), following the manufacturer’s instructions. A pair of PCR primers was designed to detect the 7-bp deletion in the sixth exon of CmDFR (Supplementary Figure S1 ). The PCR mixture contained 100 ng of genomic DNA, 5 pmol of each primer, 10 pmol of dNTPs, and 1 unit of PrimeSTAR HS DNA Polymerase in 1 × PrimeSTAR Buffer (Takara) in a total volume of 25 μL. The PCR conditions were as follows: An initial denaturation at 98 °C for 2 min, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 60 °C for 10 s, and an extension at 72 °C for 20 s, and a final incubation at 72 °C for 5 min. The PCR products were separated in a 5% nondenaturing polyacrylamide gel in 1 × TBE buffer (90 mM Tris-borate, 2 mM EDTA, pH 8.0). Following electrophoresis, the DNA fragments were visualized by silver staining, as previously described [26 (link)].
5
Cloning and Expression of Thermophilic Proteases
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Each gene was amplified by PCR from F. islandicum AW‐1 genomic DNA with appropriate primers (Table S4 ). PCR was performed in a C1000 Touch™ Thermal Cycler (Bio‐Rad) using the following conditions: 98°C for 5 min, 30 cycles at 98°C for 30 sec, 55°C for 15 sec and 72°C for 1 min kb−1, and a final extension at 72°C for 10 min. The PCR mixture (50 µl) contained 100 ng of genomic DNA, 10 pmol of each primer, 0.2 mM dNTP mix, 10 µl of 5 × PrimeSTAR buffer (Takara) and 1.25 U of PrimeSTAR polymerase. The PCR products were cloned into pTOP Blunt V2, and the resulting constructs were transformed into E. coli DH5α. Transformants containing pTOP Blunt V2 vectors harbouring protease‐encoding genes were selected on Luria–Bertani (LB) plates containing ampicillin (100 µg ml−1). Plasmid DNA was isolated from transformants, digested with restriction enzymes and ligated into the pET‐28a (+) vector. The resulting plasmids were transformed into E. coli BL21 (DE3) cells, which were then grown at 37°C in LB broth containing kanamycin (50 µg ml−1) to an absorbance at 600 nm of 0.4–0.6. After induction with 0.2 mM isopropyl‐β‐D‐thiogalactopyranoside, the cells were grown overnight at 37°C before they were harvested by centrifugation at 10 000 g for 30 min at 4°C.
6
Engineered Emerald Luciferase Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cDNA for Eluc (Emerald Luc®) was purchased from TOYOBO (Shiga, Japan). Mutations at R214, H241, S246, and H347 of Eluc were introduced by site-directed mutagenesis with the primers shown in Table 1 and their complementary primers. Template DNA (approx. 0.1 μmol) was added to a polymerase chain reaction (PCR) mixture containing 0.8 μM 5'phosphorylated primer, 0.2 mM dNTP mix, 5× PrimeSTAR Buffer, and 2.5 U of PrimeSTAR HS DNA Polymerase (Takara Bio Inc.). The PCR cycling parameters were the following: the solution was heated at 98°C for 2 min, followed by 30 cycles of 10 s at 98°C, 5 s at 55°C, and 7 min at 72°C. The mixture was treated with DpnI (10 U; Takara Bio Inc.) at 37°C for 4 h. The introductions of the mutations were verified using DNA sequencing (Eurofins Sequencing Service).
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!