Protocol This protocol requires 30 minutes hands-on processing time. 1 2 4 3 Place 50mg of ethanol-stored Penaeus vannamei into a new 1.5ml tube and incubate for 15 minutes at 37°C with the cap open. Add 200µl of Tissue Lysis Buffer (TLA) and 20µl of Proteinase K to each sample tube. Incubate at 60°C overnight, continuously mixing the sample in lysis buffer at 900rpm. Transfer the entire sample volume to well #1 of each Maxwell® 16 cartridge. 5 7 6 8 9 A. 10 20 30 40 50 60 70 80 0 QuantiFluor® dsDNA System B. 1,000 100 200 300 400 500 600 700 800 900 0 Concentration A260 /A280 A260/A230 0.50 1.00 1.50 2.00 2.50 0 NanoVue™ Plus Spectrophotometer Purified shrimp DNA (3.1ng) was amplified using GoTaq® qPCR Master Mix (Cat.# A6001) and universal primers for animal mitochondrial rRNA [from Yang, L. et al. (2014) Sci. Rep. 4, 4089] in a 50µl reaction (n = 3; Table 1). To test potential qPCR inhibition, DNA samples were diluted 1:10. An range of ΔCt (Ct value of 1:10 DNA dilution – Ct value of undiluted DNA) between 2.5 and 3.3 is accepted and it suggests that no qPCR inhibition has occurred. Conclusion Shrimp DNA (n = 3) Table 1. qPCR Analysis of Purified Shrimp DNA Undiluted DNA 1:10 DNA dilution ∆Ct (1:10—Undiluted DNA) Positive Control No-template control Ct Value 31.9 34.82 2.92 23.95 Undetermined 0.06 0.10 0.15 Standard Deviation The custom Maxwell® 16 FFS Nucleic Acid Extraction System can extract amplifiable DNA from shrimp without qPCR inhibitors. Ask your branch about the custom Maxwell® 16 FFS Nucleic Acid Extraction System » Learn more about the Maxwell® 16 System » enews | August 2015 Add 200µl of Lysis buffer to well #1 of each Maxwell® 16 cartridge. Place one of the supplied elution tubes into the sample rack and add 50µl of the supplied Elution Buffer for each sample. Place the plunger in well #8. Select LEV configuration on the Maxwell® Press Start. Results Figure 1 shows the concentration of purified shrimp DNA using two different methods. Figure 1. Purified shrimp DNA concentration measured using a fluorescent dsDNA dye and UV spectrophotometry. Panel A. Shrimp DNA purified from 50mg of muscle was quantitated using the QuantiFluor® dsDNA System (Cat. #E2670). Concentration averaged from three samples. Panel B. Shrimp DNA purified from 50mg of muscle tissue was quantitated using the NanoVue™ Plus Spectrophotometer (n = 3), and purity determined using absorbance readings at 230nm, 260nm and 2680nm. 16 Instrument and select method: RUN, DNA: Blood. DNA Concentration (ng/µl) DNA Concentration (ng/µl) Absorbance Ratio 13136MA Pagina 6
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