Ct Value 13134MA Ct Value 13135MA Protocol Both Protocols require hands-on time of 20–30 minutes. The automated purification finishes in less than 1 hour. 1 2 3 4 5 6 Place the desired number of 4mm punches (2, 4 or 8 punches for DNA; 20, 50 or 100mg tissue for RNA) into a 2ml screw-top tube. Note: One 4mm punch is approximately 2.5mg of leaf tissue. For DNA purification, add 300µl of Tissue Lysis Buffer (TLA) and 10µl of RNase A solution to each sample tube. For RNA purification, add 400µl of chilled 1-Thioglycerol/ Homogenization Solution to each sample tube. Homogenize samples for desired time using a bead-beating device (e.g., FastPrep® -24 Instrument at 6M/S, twice for 40 seconds with 20 second delay between each time using 4mm 440C stainless steel grinding balls). For RNA purification, add 200µl of Lysis Buffer to the homogenate, and vortex for 15 seconds to mix. Incubate samples at room temperature for 10 minutes. Centrifuge samples in a microcentrifuge at maximum speed for 2 minutes. For DNA purification, add 300µl of Nuclease Free Water to well #1 of each Maxwell® 16 Plant cartridge. Conclusion The Maxwell® 16 Plant DNA and RNA Kits on the Maxwell® 16 Instrument result in high-quality, amplifiable nucleic acids suitable for use in downstream analysis such as real-time PCR. 15 16 17 18 19 20 21 24 82 48 24 8 Brassica juncea (Mustard green) Brassica juncea (Mustard red) Number of Leaf Punches Figure 1. Purified DNA isolated from 2, 4 or 8 Brassica leaf punches was analyzed by qPCR. One microliter of eluted DNA was amplified using the GoTaq® qPCR Master Mix (Cat.# A6001) and 10µM Plant Universal Primers [from Wang, J. et al. (2011) Plant Methods 7, 39] in a 25µl reaction. Brassica napus (red Russian kale) 20.0 20.5 21.0 21.5 22.0 22.5 23.0 23.5 24.0 24.5 25.0 20 50 1002050100 Brassica juncea (Mustard green) Brassica juncea (Mustard red) Leaf Tissue (mg) Figure 2. Purified RNA isolated from 20, 50 or 100mg of Brassica leaf tissue was analyzed by RT-qPCR. One microliter of eluted RNA was amplified using the GoTaq® 1-Step RT-qPCR System (Cat.# A6020) with PP2A primers [from Wang, Z. et al. (2014) Molecular Genetics and Genomics 289, 1023–35.] in a 20µl reaction. All graph bars are mean ± standard deviation of n = 3. Learn More about Maxwell® 16 LEV Plant DNA Kit » Learn More about Maxwell® 16 LEV Plant RNA Kit » enews | August 2015 20 50 100 Brassica napus (red Russian kale) 7 8 9 10 11 12 Transfer the entire volume of supernatant to well #1 of the Maxwell® 16 Plant cartridge. For RNA purification, add 5µl of DNase to well #4. 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® 16 Instrument, and choose the appropriate extraction method: RUN, DNA: Plant for DNA purification or RUN, RNA: Plant for RNA purification. Press start. Pagina 2
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