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Download BiotoolTM Plant Leaf Direct PCR Kit (B4003)
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Transcript
BiotoolTM Plant Leaf Direct PCR Kit (B4003) Introduction The BiotoolTM Plant Leaf Direct PCR Kit provides a fast one-step preparation and PCR amplification that is specifically designed for multiple plants excluding the polysaccharide and polyphenols plants. Biotool’s trademarked Buffers rapidly digest plant leaves to release intact genomic DNA that can be used directly as template for PCR amplification. By using this kit, the genomic DNA extractions can be done within 10-15 minutes. In addition, the Biotool 2x P-PCR OPTITM Mix (which includes an optimized Taq Polymerase) ensures high amplification efficiency of target DNA. Buffer B 10 minutes PCR 95 oC 2x P-PCR OPTITM Mix Buffer A Application Direct PCR of plant leaves including rice, wheat, maize, rapeseed plant, soybean and other plants. The BiotoolTM Plant Leaf Direct PCR plus Kit is available for polysaccharide and polyphenols plants including arabidopsis, cotton, banana, potato, tomato, etc. Kit Contents Catalog # B40023 (200 rxns) B40025 (500 rxns) B40028 (2000 rxns) Buffer A (mL) 10 25 100 Buffer B (mL) 10 25 100 2x P-PCR OPTITM Mix (mL) 2 5 20 6x DNA Loading Buffer (mL) 0.6 1.5 6 User Guide Yes Yes Yes Contents Buffer A: Lysis buffer Buffer B: Neutralization buffer 2x P-PCR OPTITM Mix: Includes Biotool’s trademarked and optimized Taq DNA polymerase, dNTPs, MgCl2, and reaction buffer. 6x DNA Loading Buffer: We strongly recommended this loading buffer in agarose gel electrophoresis. Storage 2x P-PCR OPTITM Mix should be stored at -20 ℃. If the mix is to be used frequently, it can be stored at 4 ℃ for up to 10 days. Buffers can be stored at 4 ℃ for up to 2 years. Experimental Protocol 1. Genomic DNA Preparation Place plant leaf sample (5-10 mg, diameter 5-7 mm) in a centrifuge tube. Add 50μL of Buffer A (be sure to submerge the entire sample). Incubate the tube at 95 ℃ for 10 minutes. Add 50μL of Buffer B and vortex briefly. 2. PCR Add primers and template into 2x P-PCR OPTITM Mix according to the recommended concentrations. Give the mixture a quick spin in the centrifuge and load into PCR amplifier to begin amplification. PCR Reaction Components 20 μL Reaction Volume (μL) 50 μL Reaction Volume (μL) 2x P-PCR OPTI 10 25 Forward Primer (10 μM) 0.5 1 Reverse Primer (10 μM) 0.5 1 Template 2 5 H2O 7 18 Total Reaction Volume 20 50 Temperature (℃) Time Cycles 94 5 min 1 94 20 sec 45-65 30 sec 72 X min (1kb/min) 72 5 min 1 12 -- 1 TM Mix 30-40 Note: Before First Use Avoid repeat freeze-thaw cycles of the 2x P-PCR OPTITM Mix. Please note that if the mixture turns viscous (especially at high temperature), simply incubate on ice for 1-2 minutes, and then gently shake 3-5 times prior to using. Trouble Shooting Problem No amplification product in test or control samples Amplification worked in the control samples, but not in test samples Non-specific amplification product (s) Over-exposed signal on agarose gel Potential Cause(s) Suggestion(s) Amplification reaction was incorrectly set up Optimize the proper reaction set up Improper storage has led to loss of activity of PCR reagents Replace all components with fresh reagents Primers are not optimal and did not anneal Redesign primers Digestion was incomplete Extend digestion time up to 20 minutes at 95℃ Lysis solution was stored too long and genomic DNA degraded Collect fresh plant leaf samples for genomic DNA extraction Unknown contaminants interfered with amplification reaction Dilute template in purified H2O or TE buffer and repeat PCR The quantity of the amplification product was not sufficient Increase the number of PCR cycles to 35-40 to yield more amplification product Annealing temperature too low Increase the annealing temperature The number of PCR cycles was too high Decrease the number of cycles to 30-35 Primer concentration was too high Decrease primer concentration Template concentration was too high Dilute template in purified H2O or TE buffer Loading dye may be sub-optimal for your specific reaction product Switch to a different brand of loading dye