In fecal, serum and ovarian samples, avian HEV RNA was tested according to previously described methods (Zhao et al.,2017 (link)). Briefly, RNA was extracted from 200 μL sera or 10 % fecal or tissue suspension using TRIzol reagent in accordance with the manufacturer's instructions. For RT-nPCR, reverse transcription and first PCR were conducted using PrimeScript One Step RT-PCR Kit (TaKaRa, Osaka, Japan). Next, TransTaq High Fidelity DNA polymerase (TransGen Biotech Co., Beijing, China) was used for the second PCR in accordance with the manufacturer's instructions. The final PCR products were electrophoresed on a 1% agarose gel.
Transtaq high fidelity dna polymerase
Manufactured by Transgene
Sourced in China
TransTaq High Fidelity DNA polymerase is a thermostable DNA polymerase used for high-fidelity DNA amplification. It exhibits 3'-5' exonuclease activity, providing proofreading capability to improve the accuracy of DNA synthesis.
Automatically generated - may contain errors
Lab products found in correlation
One step primescript rt pcr kit, by Takara Bio (3 mentions)
Tianamp genomic dna kit, by Tiangen Biotech (2 mentions)
Transzol reagent, by Transgene (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
Pmd19 t vector system, by Takara Bio (1 mentions)
Dna clean concentrator 25, by Zymo Research (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Peasy t5 zero cloning kit, by Transgene (1 mentions)
Universal dna purification kit, by Tiangen Biotech (1 mentions)
8 protocols using transtaq high fidelity dna polymerase
1
Avian Hepatitis E Virus Detection
2
Detecting Swine HEV RNA in Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Swine HEV RNA in fecal and serum samples was detected using RT-nPCR as described previously (Huang et al., 2002 (link)). Specifically, viral RNA was extracted from 200 μl sera or 10% fecal volume using TransZol Reagent (TransGen, China) according to the manufacturer’s instructions. Reverse transcription and the first PCR were performed for RT-nPCR with the PrimeScript™ One Step RT-PCR Kit (Takara, Tokyo, Japan), and the second PCR was performed with TransTaq High Fidelity DNA polymerase (TransGen, Beijing, China). Finally, the PCR products were analyzed with gel electrophoresis.
3
HEV Detection in Fecal Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
200 μL of 10 % fecal suspension was used for total RNA extraction by TRIzol Reagent (TaKaRa, China). All samples were analyzed using a broad-spectrum nested reverse transcription polymerase chain reaction (RT-nPCR) with specific primers designed to amplify the partial RdRp region of HEV ORF1 gene, which were described previously by Reimar Johne [26 (link)]. In addition, to confirm detection of HEV, the partial ORF2 gene of the HEV genome was also amplified using RT-nPCR as described previously [27 (link)]. Briefly, for RT-nPCR, reverse transcription and first PCR were performed using PrimeScript™ One Step RT-PCR Kit (TaKaRa, China). Next, the second PCR was conducted using TransTaq High Fidelity DNA polymerase (TransGen Biotech, China) based on the manufacturer’s instructions. Finally, PCR products were identified by electrophoresis on 1% agarose gel. To avoid cross-contamination, the negative controls were set up in all of these experiments, and filter tips were also used throughout the process. All purified positive PCR products were sequenced by Genetic Analyzer(ABI 3130, Applied Biosystems, USA). Two sequences were submitted to GenBank (Accession numbers MW498242 and MW498243).
4
PCV2 Genome Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Eighteen samples from eight farms were determined to be positive for PCV2. These samples were used for genome amplification and sequencing using a method described in the literature (Huang et al., 2013) . The complete sequence was amplified with 5'-TAGCCGCGGG CTGGCTGAACTTTTGA-3' (sense primer) and 5'-AGACCGCGGAAATTTCTGACAAAC G-3' (reverse primer). A reaction mixture of 25 μL contained 2.5 μL 10X PCR buffer, 2 μL dNTP mix (2.5 mM each), 1 μL of primers (10 pmol/μL), 1.5 μL DNA, 0.25 μL TransTaq ® High Fidelity DNA Polymerase (TransGen, China), and double-distilled water. The PCR con-ditions were 94°C for 3 min; followed by 30 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 2 min; and a 10-min extension at 72°C. The PCR products obtained were separated by 1% agarose electrophoresis, extracted with an E.Z.N.A. ® Gel Extraction Kit (OMEGA), and ligated with a PMD19-T vector system (Takara, Japan). The recombinant plasmids were transformed into Escherichia coli DH5α-competent cells and then sequenced in positive clones by Invitrogen (Guangzhou, China). A consensus sequence was obtained for each genomic DNA using a method described in the literature (Cai et al., 2012) .
5
Amplification of Cotton GbD14 Gene
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cDNA sequence of the GbD14 gene was obtained from the cotton database (https://www.cottongen.org/blast ) alignments, and specific primers (GbD14F: 5′-ATGGCAAACACCCTTTTAGAAG-3′; GbD14R: 5′-CTAGAAAACTCACCGCGGAAG-3′) were designed using the software Primer Premier 6.0. Using the cDNA of Xinhai 15 as a template, we amplified the target GbD14 sequence via TransTaq DNA Polymerase High Fidelity (TransGen, Beijing, China).
6
Genome-wide CRISPR perturbation screening
7
CRISPR-Mediated Gene Editing Validation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For the validation of critical substitutions of PSMB5 and PLK1, sgRNAs were designed near the mutation sites, and each 119-nt ssODN donor encoded one amino acid substitution for a validated residue. All sgRNAs and ssODN donor sequences are listed in Additional file 5 : Table S8. HeLa cells were transfected with 1 μg of sgRNA and 2 μg of the ssODN donor in six-well plates. Fourteen days post-transfection, 1.5 × 105 cells were seeded in six-well plates 24 h before drug selection. Cells were treated with corresponding drugs at the proper dosages for 72 h: Bortezomib (10 ng/ml) and BI2536 (10 ng/ml). Genomic DNAs of drug-resistant cells were extracted using TIANamp Genomic DNA Kit (TIANGEN). The mutated loci were amplified using TransTaq DNA Polymerase High Fidelity (Transgen) and purified using a Universal DNA Purification Kit (TIANGEN). Primers are listed in Additional file 5 : Table S9. PCR fragments were cloned into pEASY-T5 Zero Cloning Kit (Transgen) for sequencing.
8
Viral DNA Extraction and Amplification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The viral DNA was extracted from purified virus particles using TIANamp Genomic DNA Kit (TIANGEN). According to the reported genome sequence of HaDV2 (GenBank accsession No.: HQ613271), three forward primers near the 3′ end (DVF1 [nt 4576–4595], DVF2 [nt 3891–3910], DVF3 [nt 4343–4362]), and two reverse primers near the 5′ end (DVR1 [nt 1038–1057] and DVR2 [nt 832–889]) were designed according to the genome sequence of HaDV2 (Additional file 1 : Table S1). PCR reactions were performed using TransTaq DNA Polymerase High Fidelity (TransGen, Beijing, China) and extracted viral DNA as a template. The PCR program was as follows: 30 s at 94 °C, 30 s at 57 °C, and 60 s at 72 °C for 40 cycles.
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!