Minibest plasmid extraction kit ver 5

Manufactured by Takara Bio

Sourced in China

The MiniBEST Plasmid Extraction Kit Ver.5.0 is a laboratory equipment product designed for the quick and efficient extraction of plasmid DNA from bacterial cultures. The kit provides a simple and reliable method for isolating high-quality plasmid DNA suitable for various downstream applications.

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Lab products found in correlation

Pmd18 t vector, by Takara Bio (3 mentions) Escherichia coli dh5α competent cells, by Takara Bio (1 mentions) Dh5α competent cells, by Takara Bio (1 mentions) One step primescript rt pcr kit, by Takara Bio (1 mentions) Minibest dna fragment purification kit ver 4, by Takara Bio (1 mentions) Escherichia coli e coli dh5α competent cells, by Takara Bio (1 mentions) Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)

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Plasmid extraction kit (19 citations)

3 protocols using minibest plasmid extraction kit ver 5

Plasmid-based Molecular Standards for ASFV, CSFV, and PRRSV

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The total DNA was extracted from the ASFV positive sample, and the total RNA was extracted from CSFV and PRRSV vaccine viruses and then reverse transcribed to cDNA. The target fragments of ASFV, CSFV, and PRRSV were amplified by PCR using ASFV DNA and CSFV, PRRSV cDNA as templates. The amplicons were purified and cloned into the pMD18-T vector (TaKaRa) and transferred to Escherichia coli DH5α competent cells (TaKaRa). The positive clones were cultured at 37°C for 18 h–20 h and extracted by MiniBEST Plasmid Extraction Kit Ver.5.0 (TaKaRa) for the plasmid constructs. The plasmids were called p-ASFV, p-CSFV, and p-PRRSV, respectively, and stored at −20°C until used as the standard plasmids.
The standard plasmids were quantified by ultraviolet absorbance at 260 nm and 280 nm using a NanoDrop spectrophotometer (Thermo Fisher, USA). Their concentrations were calculated as follows: plasmid copy number (copies/μL) = (plasmid concentration × 10−⁹ × 6.02 × 10²³)/(660 Dalton/bases × DNA length).

Chen Y., Shi K., Liu H., Yin Y., Zhao J., Long F., Lu W, & Si H. (2021). Development of a multiplex qRT-PCR assay for detection of African swine fever virus, classical swine fever virus and porcine reproductive and respiratory syndrome virus. Journal of Veterinary Science, 22(6), e87.

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Molecular Detection of Swine Viral Pathogens

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RNA/DNA was extracted from a clinical PRCoV-positive specimen or a PRRSV, PRV, and SIV vaccine solution and was used as a template to amplify PRCoV, PRRSV, SIV, and PRV gene fragments via PCR using the primers in Table 1 with a One Step PrimeScript™ RT-PCR Kit (Perfect Real Time) (TaKaRa, Dalian, China). The PCR products were purified using a MiniBEST DNA Fragment Purification Kit Ver.4.0 (TaKaRa, Dalian, China), cloned into a pMD18-T vector (TaKaRa, Dalian, China), and transformed into DH5α competent cells (TaKaRa, Dalian, China). Positive clones were cultured at 37 °C overnight for 20–24 h, and plasmid constructs were extracted using a MiniBEST Plasmid Extraction Kit Ver.5.0 (TaKaRa, Dalian, China). The four standard plasmid constructs were named p-PRCoV, p-PRRSV, p-SIV, and p-PRV. The OD260/OD280 nm values of the standard plasmid constructs were measured, and their concentrations were determined using the following formula:

P l a s m i d s (c o p i e s / μ L) = \frac{6.02 \times 10^{23} \times p l a s m i d c o n c e n t r a t i o n \times 10^{- 9}}{p l a s m i d l e n g t h (b p) \times 660}

Ma Y., Shi K., Chen Z., Shi Y., Zhou Q., Mo S., Wei H., Hu L, & Mo M. (2024). Simultaneous Detection of Porcine Respiratory Coronavirus, Porcine Reproductive and Respiratory Syndrome Virus, Swine Influenza Virus, and Pseudorabies Virus via Quadruplex One-Step RT-qPCR. Pathogens, 13(4), 341.

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Recombinant Plasmid Generation for PRRSV Strains

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The total nucleic acids were extracted from vaccine strains of C-PRRSV, HP-PRRSV, and the positive sample of NL-PRRSV, reverse transcribed to cDNA, and then amplified by PCR using the specific primers. The amplicons were purified and cloned into pMD18-T vector (TaKaRa, Dalian, China) and transferred into Escherichia coli (E. coli) DH5α competent cells (TaKaRa, Dalian, China). The positive clones were cultured at 37 °C for 22–24 h and extracted by MiniBEST Plasmid Extraction Kit Ver.5.0 (TaKaRa, Dalian, China) for plasmid constructs. The recombinant plasmids were named p-C-PRRSV, p-HP-PRRSV, and p-NL-PRRSV and stored at −70 °C until used as standard plasmids.
The standard plasmids were quantified by ultraviolet absorbance at 260 nm and 280 nm with a NanoDrop spectrophotometer (Thermo Fisher, Waltham, MA, USA). Their concentrations were calculated according to the following formula: plasmid copy number (copies/μL) = (6.02 × 10²³ × plasmid concentration × 10^–9)/(660 × plasmid length).

Long F., Chen Y., Shi K., Yin Y., Feng S, & Si H. (2023). Development of a Multiplex Crystal Digital RT-PCR for Differential Detection of Classical, Highly Pathogenic, and NADC30-like Porcine Reproductive and Respiratory Syndrome Virus. Animals : an Open Access Journal from MDPI, 13(4), 594.

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