The MCPH1 gene targeted regions were amplified using primers MCPH1-E2-5939bp-f: 5′- GGCGGGGGGATAACGGTGCCGAAAG-3′. MCPH1-E2-5939bp-r: 5′-GACAGGCATTAGGGAGGTCAAACAAGGCTCTTAGGGTA-3′ and MCPH1-E4-5713bp-f: 5′-GTTTTCAAGGTTCATCATGTTGTCATCTGTATT-3′MCPH1-E4-5713bp-r: 5′-ATTGTTTATGATTAGTGAGACGAAGGATTTGC-3′. The PCR was performed using LA Taq DNA Polymerase (ClonTech). We prepared libraries following the PacBio protocol, every five PCR products (with distinct barcode) are pooled together, DNA damage repair, EXO III and VII digestion, Two AMPure PB bead washes, annealing, binding, and sequencing.
La taq dna polymerase
Manufactured by Takara Bio
Sourced in Japan, China, United States
LA Taq DNA polymerase is a thermostable DNA polymerase enzyme used for PCR amplification. It exhibits high processivity and fidelity in DNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
Dna purification kit, by Takara Bio (12 mentions)
Restriction endonuclease, by Takara Bio (12 mentions)
T4 dna ligase, by New England Biolabs (10 mentions)
Pgem t easy vector, by Promega (10 mentions)
T4 dna ligase, by Takara Bio (7 mentions)
Pmd18 t vector, by Takara Bio (6 mentions)
Qiaquick gel extraction kit, by Qiagen (5 mentions)
Beechwood xylan, by Merck Group (5 mentions)
Restriction endonuclease, by New England Biolabs (5 mentions)
Primescript 2 1st strand cdna synthesis kit, by Takara Bio (4 mentions)
P pastoris gs115, by Thermo Fisher Scientific (4 mentions)
Pmd19 t vector, by Takara Bio (4 mentions)
Total rna isolation system kit, by Promega (4 mentions)
T4 dna ligase, by Promega (4 mentions)
Rnaiso plus, by Takara Bio (3 mentions)
Superscript 3 reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Barley β glucan, by Merck Group (3 mentions)
Ppic9, by Thermo Fisher Scientific (3 mentions)
Pgem t easy, by Promega (3 mentions)
183 protocols using la taq dna polymerase
1
MCPH1 Gene Amplification and Sequencing
2
Genotyping of Meprin Gene Knockouts
3
Transcriptome Analysis of PBMC Response to rHE4
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5 × 107 PBMCs from single donor were suspended in 5 mL of serum free RPMI1640 medium (Invitrogen, 31800) and incubated with or without 0.01 μg/mL of rHE4 (Abcam, ab184603) for 6 h, and total RNA was isolated using TRIzol™ Reagent (Invitrogen, 15596018). Next, mRNA was purified using Magnosphere™ UltraPure mRNA Purification Kit (Takara-Clontech, 9186). From the 5 μg of mRNA, subtractive cDNA libraries were constructed using PCR-Select™ cDNA Subtraction Kit (Takara-Clontech, 637401) following the manufacturer's protocols (Figure S1A ). PCR products of the differentially expressed genes were cloned into a pUC19-TA vector. Top 10 competent cells (Invitrogen, C404003) were transformed with the clones and were seeded on Xgal/IPTG containing LB/ampicillin plates. The colonies of clones containing the inserts were selected by blue/white selection and were amplified by direct colony PCR using LA Taq® DNA polymerase (Takara-Clontech, RR002A) and M13 primers (Table S1 ). PCR products in the range of 200 to 3000 bp were then subjected to direct sequencing (Figures S1B,C ).
4
cDNA Synthesis and HvSAMS3 Amplification
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First-strand cDNA was synthesized according to Kim [39 (link)] from total RNA using a RevertAid™ (Thermo Scientific, Waltham, MA, USA). For PCR amplification, the first Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) was used as a template. PCR amplification of HvSAMS3 was performed using LA-Taq™ DNA Polymerase (Takara, Beijing, China). The amplified PCR fragments were recovered, cloned into the pMD19-T vector using a TA cloning kit (Takara, Beijing, China), and sequenced. The primers to clone HvSAMS3 are presented in Table S2 .
5
Cucurbitaceae Leaf Transcriptome Analysis
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Plants of the Cucurbitaceae family were cultivated in a natural environment. Leaves of all Cucurbitaceae plants were collected in the summers of 2014 and 2015 from the Guangxi University of Chinese Medicine (Momordica cochinchinensis, Trichosanthes rubriflos, Trichosanthes truncata, and Trichosanthes kirilowii) and from the Guangxi Medical University (Gynostemma pentaphyllum, Momordica charantia, Benincasa hispida var. chieh-qua, Luffa acutangula, and Cucurbita moschata) in Guangxi Province, China. Fresh leaves were sampled, immediately frozen in liquid nitrogen, and stored at -80°C for RNA isolation. All the samples were authenticated by Prof. Ruisong Huang and Prof. Yaosheng Wu. The kits used in the experiments were as follows: Takara RNAiso Plus™ (Code No.: 9109), Takara PrimeScript™ II 1st Strand cDNA Synthesis Kit (Code No.: 6210A), Takara LA Taq DNA polymerase (Code No.: RR02MA), Takara 3′-Full RACE Core Set with PrimeScript™ RTase (Code No.: 6106), Takara MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 (Code No.: 9762), Takara MiniBEST DNA Fragment Purification Kit Ver.4.0 (Code No.: 9761), Takara Premix Taq™ Version 2.0 plus dye (Code No.: RR901A), and TransGen pEASY-T1 cloning kit (Code No.: CT101-02). Primers were synthesized by the IGE Biotech Company (Guangzhou City, China).
6
Quantitative Expression Analysis of Rice Genes
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Total RNA was first extracted from rice plants with Trizol method, and then first-strand cDNA was synthesized using a Superscript III Reverse Transcription Kit (Invitrogen, Carlsbad, CA, USA). Semiquantitative PCR was performed using LA Taq DNA polymerase (TaKaRa) with the rice ACTIN1 gene serving as an internal control. The PCR program included 1 min at 94 °C followed by 30 cycles at 94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 10 min. Quantitative PCR was performed using a SYBR Premix Ex Taq2 kit (TaKaRa) on ABI PRISM 7900HT under the following conditions: 10 s denaturing at 95 °C, 30 s annealing at 60 °C, 40 cycles. The mRNA amount relative to ACTIN1 was finally calculated. Specific primers were shown in Table S4 .
7
Marker-free Piglet Genotyping via PCR
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To detect marker-free piglets, genomic DNA was obtained from ear tissue using phenol-chloroform extraction. Genomic DNA (100 ng per reaction) was subjected to PCR analysis using LA-Taq DNA polymerase (TaKaRa, Dalian, China, TaKaRa Code: RR52A) and appropriate primers (S1 Table ). PCR products were analyzed by agarose gel electrophoresis. For sequencing analysis, PCR products corresponding to marker-free fragments were purified using QIAquick Gel Extraction Kit (QIAGEN, Germany, Catalogue: 28704) and cloned into the pMD-19T (TaKaRa, Dalian, China, TaKaRa Code: 6013). The cloned plasmids were then sequenced using M13 primers.
8
Medaka PTEN Gene Expression Analysis
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Total RNA was isolated from medaka embryos with the use of an RNeasy Mini Kit (Qiagen, Hilden, Germany) and an RNase-Free DNase Set (Qiagen) in order to eliminate genomic DNA from the samples. The extracted RNA was subjected to RT with the use of a Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Rotkreuz, Switzerland), and the presence of medaka pten cDNAs among the RT products was determined with a medaka ptena primer set (ptena-S, 5'-ATGGTCAGTCGGAACAAAAGGAGATAC-3' ; ptena-E, 5'-CACTTTTGTGATTTGCGTGTGCTGCTC-3' ) and a medaka ptenb primer set (ptenb-S, 5'-ATGGCCGCCAATCTAATCAAGGAGATCGTG-3' ; ptenb-E, 5'-ATCTGTTCGTGGTCGTCCTCCTC-3' ). The amplification protocol comprised an initial incubation at 94°C for 1 min; 35 cycles of 98°C for 10 s, 56°C for 20 s, and 72°C for 75 s; and a final incubation at 72°C for 7 min. The reaction mixture consisted of 2 μl of 10×LA buffer (Takara, Kyoto, Japan), 0.5 μl of cDNA, 0.5 μM of each primer, and 0.5 U of LA Taq DNA Polymerase (Takara) in a total volume of 20 μl.
9
Characterizing Rice OsCAS Calcium Signaling
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Total RNA of rice seedling leaves was extracted using Trizol reagent (Invitrogen Inc., Gaithersburg, MD, USA). After RNase-free DNase I treatment, 1 μg of total RNA was used to synthesize cDNA using SuperScript III reverse transcriptase (TaKaRa Bio Inc., Dalian, China). Specific primers were used in a polymerase chain reaction (PCR) to amplify the 1164-bp full-length cDNA of OsCAS by using LA-Taq DNA Polymerase (TaKaRa), according to the manufacturer’s protocols. Primer pairs (1) and (2) used for PCR, designed using Primer Premier 5.0, are shown in S1 Table . PCR products were purified and cloned into a pcDNA3 vector. HEK293 cells were then transiently transfected with empty pcDNA3 vector, OsCAS, and Arabidopsis CAS. Transfected cells were screened for CICI (Ca2+o-induced [Ca2+]i increases) using ratiometric imaging of the fluorescent Ca2+-sensitive dye Fura-2 [21 (link)].
10
Recombinant Glycoside Hydrolase Expression
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Strain A. fumigatus Af293 was obtained from the Fungal Genetics Stock Center, which genome has been published in 2005. Escherichia coli Trans1-T1 (TransGen Biotech, Beijing, China) was cultivated in Luria-Bertani (LB) medium at 37°C for gene cloning and sequencing. P. pastoris GS115 (Invitrogen, Carlsbad, CA) cultivated in yeast peptone dextrose (YPD) medium at 30°C was used for gene expression. The plasmids pGEM-T Easy (Promega, Madison, WI) and pPIC9 (Invitrogen) were used as cloning and expression vectors, respectively. Beechwood xylan, barley β-glucan, carboxymethyl cellulose sodium (CMC-Na) and locust bean gum were purchased from Sigma-Aldrich (St. Louis, MO). Soluble wheat arabinoxylan was obtained from Megazyme (Wicklow, Ireland). The DNA purification kit, LA Taq DNA polymerase and restriction endonucleases were purchased from TaKaRa (Otsu, Japan). SV Total RNA Isolation System was purchased from Promega.T4 DNA ligase was from New England Biolabs (Hitchin, UK). All chemicals were of analytical grade and commercially available.
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