Cellular total RNA was extracted with Trizol reagent (Thermo Fisher Scientific) and cDNA templates were generated via RT-PCR using high-capacity RNA-to-cDNA kits (Applied Biosystems, Carlsbad, CA, USA). PCR was performed with 2 µL synthesized cDNA using an LA PCR Kit (Takara Bio, Kusatsu, Japan), and the housekeeping gene GAPDH was employed as the internal control. PCR products were detected by 1% agarosegel electrophoresis. The primers of AFP and GAPDH were: AFP forward, 5′-TCAGTGAGGACAAACTATTGG-3′; AFP reverse, 5′-CTCTTCAGCAAAGCAGACTTC-3′; GAPDH forward, 5′-GCACCACCAACTGCTTAGC-3′; and GAPDH reverse, 5′-GGCATGGACTGTGGTCATA-3′.
La pcr kit
Manufactured by Takara Bio
Sourced in Japan
The LA PCR Kit is a laboratory equipment product designed for the amplification of long and accurate DNA fragments. It provides reagents and protocols for performing high-fidelity polymerase chain reaction (PCR) experiments.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
High capacity rna to cdna kit, by Thermo Fisher Scientific (1 mentions)
Abi 3730 automated dna sequencer, by Thermo Fisher Scientific (1 mentions)
Peasy t1 vector, by Transgene (1 mentions)
Easypure pcr purification kit, by Transgene (1 mentions)
Abi 3500xl capillary dna analyzer, by Thermo Fisher Scientific (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Dneasy kit, by Takara Bio (1 mentions)
Nextera xt dna sample preparation kit, by Illumina (1 mentions)
Miseq reagent kit v2, by Illumina (1 mentions)
7 protocols using la pcr kit
1
Quantification of AFP Gene Expression
2
Genetic Analysis of Phloem Feeding Insect
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Adult specimens of P. tibialis were collected from alfalfa in Yuzhong County, Lanzhou City, Gansu Province, China, on 5 June 2014. Samples and voucher specimens are deposited in the State Key Laboratory of Grassland Agro-Ecosystem, College of Pastoral Agricultural Science and Technology, Lanzhou University in Lanzhou, China. All specimens were initially preserved in 100% ethanol in the field, and transferred to −20 °C until used for DNA extraction. The total genomic DNA was extracted from thorax muscle of a single specimen using the insect genome DNA extraction kit (Omega, Norcross, GA, USA), according to the manufacturer’s protocols. Polymerase chain reactions (PCRs) were performed with the LA PCR Kit (TaKaRa, Shiga, Japan) following the manufacturer’s recommendations. The primers and their sources are presented in Table S1 . PCR products were electrophoresed in 1.5% agarose gel, purified with an EasyPure PCR Purification Kit (TransGen Biotech, Beijing, China), and then both strands were sequenced with primer walking on an ABI 3730 automated DNA sequencer (Applied Biosystems, Carlsbad, CA, USA).
Failed and unsatisfactory sequencing fragments were cloned into the pEASY-T1 vector (TransGen Biotech, Beijing, China), and then conducted bidirectional sequencing.
Failed and unsatisfactory sequencing fragments were cloned into the pEASY-T1 vector (TransGen Biotech, Beijing, China), and then conducted bidirectional sequencing.
3
Long-range PCR and Sanger sequencing
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Using the primers listed in Table S4 , we performed long-range PCR on 200 ng of genomic DNA. LA PCR kit (Takara) was used for these reactions and reactions were performed according to the manufacturer's protocols. Two-step PCR was performed with annealing and extension at 68°C for 20 minutes. Products were run on gels and then gel purified and sequenced by Sanger sequencing.
4
Long-range PCR characterization of EPCAM and MSH2 deletions
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A series of long-range PCR experiments designed to span the putative deletion region were performed to characterize the exact breakpoints in the EPCAM and MSH2 genes using a TaKaRa LA PCR Kit (Takara, Japan) according to the manufacturer’s protocol. The PCR products were analyzed by electrophoresis on ethidium bromide-stained 1% agarose gels and then subjected to UV detection. The expected fragment was purified and sequenced on an ABI 3500xl capillary DNA analyzer (Applied Biosystems). Details of the PCR primers are provided in Table S2
5
Quantification of DA Neuronal Markers
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To compare the alteration in DA neuronal marker and relative factors of differentiation into DA neurons, total RNA of cells was isolated using Trizol reagent (Invitrogen) and reverse-transcribed to synthesize cDNA with a RevertAidTM First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania). The LA PCR Kit (Takara, DaLian, China) was used to detect the expression of TH, DAT, Nurr1, Ptx3, and Shh mRNAs. The primer sequences are listed in Table 1 .
6
Sry, TESCO, and Sf1 Gene Sequencing
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No live animals were used in this study. Genomic DNA was extracted from tissue of an XY individual of M. minutoides or M. mattheyi (specimens collected under permits 2003/PFHG/05/GUI, 1155 MDCS/CAB-1/kss14 (link)) using a Qiagen DNeasy kit and subject to PCR amplification (Takara LA PCR kit) with primers designed based on M. musculus sequence. Primer sequences are provided in Supplementary Table S1 . Eight (for Sry), four (for TESCO), or three (for each Sf1 coding exon) independent clones from each PCR product were sequenced. Sequences were aligned using ClustalW61 (link). Identity scores were calculated using GeneStream II62 (link).
7
Whole Mitochondrial DNA Sequencing from Transgenic Mice
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Whole mtDNA was amplified from 8 Endog+/+ and 8 Endog-/- mice in one amplicon by long range PCR (Takara LA PCR kit) using 50–100 ng of DNA and specific mtDNA primers (F:5’-GGTTCGTTTGTTCAACGATTAAAGTCCTACGTG-3’, R: 5’-GAGGTGATGTTTTTGGTAAACAGGCGGGGT-3’). Quantification of amplified mtDNA was performed using Qubit 2.0 Fluorimeter (Qubit dsDNA BR Assay kit; Invitrogen). Sample libraries were prepared with Nextera XT DNA Sample Preparation kit (Illumina) according to the manufacturer's protocol for fragments to 150pb and pair-end runs. One ng of amplified mtDNA was used to prepare each library. After PCR clean up with Ampure beads XT (Beackman Coulter), libraries were normalized and pooled. Pooled libraries were carried out into the MiSeq Reagent kit V2 (300 cycles and 2×150 chemistries) (Illumina) and sequenced in the MiSeq platform and analyzed using MiSEq Control Software and MiSeq Reporter Software. Libraries were multiplexed to obtain 3000× medium coverage. The reference sequence was from Mus musculus C57BL/6 J strain (GenBank Ref. NC_005089.1 ).
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