| Description | Application：DNA amplification from plant leaves or other tender tissues; genotyping; genotype analysis of transgenic plants.Inactivation or inhibition: Activities of PlantTaq™ DNA Polymerase contained in this product can be inactivated by phenol-chloroform extraction.Inactivation or Application：DNA amplification from plant leaves or other tender tissues; genotyping; genotype analysis of transgenic plants.Inactivation or inhibition: Activities of PlantTaq™ DNA Polymerase contained in this product can be inactivated by phenol-chloroform extraction.Inactivation or inhibition：Activities of PlantTaq™ DNA Polymerase contained in this product can be inactivated by phenol-chloroform extraction.Precautions：Because the PCR reaction is extremely sensitive, contamination must be avoided during the preparation of PCR reactions. Negative control without templates is recommended for all PCR assays to control contamination.When setting up PCR reactions, 0.1-1mm and 0.3-3mm diameter leaves or other tender plant tissues of similar size are recommended for 20µl and 50µl of PCR reactions, respectively. Fresh and tender seeds are suitable for this product, but not dry and hard seeds.Instructions for Use：1. Prepare PCR reactions:a. Thaw the Easy-Load™ Plant Direct PCR Master Mix (2X) and Control Primer mix (10µM each) at room temperature. Mix well gently by inversion and centrifuge briefly. Keep the reagents on ice.b. Assemble PCR reactions on ice as follows:ComponentVolume (µl)Volume (µl)Final ConcentrationNuclease-Free Water9µl22.5µl-Easy-Load™ Plant Direct PCR Master Mix (2X)10µl25µl1XPCR Primer Mix (10µM each)1µl2.5µl0.5µM eachPlant Samples0.1-1mm diameter0.3-3mm diameter-Total Volume2050-Note 1: When setting up PCR reactions, 0.1-1mm and 0.3-3mm diameter leaves or other tender plant tissues of similar size are recommended for 20µl and 50µl of PCR reactions, respectively. Fresh and tender seeds are suitable for this product, but not dry and hard seeds. Use a clean scalpel to remove the seed shell, cut out 0.5-2mm diameter tissue, and put it directly into PCR reactions. One to two small seeds such as tomato seeds can be added directly into PCR reactions for DNA amplification. In a 50µl of PCR reaction, the diameter of the plant leaf or seed should not exceed 3mm, as too many PCR inhibitors introduced by plant samples will inhibit PCR amplification. It is recommended to perform two parallel PCR reactions for the same sample to reduce variations in sampling. To ensure the uniformity of sampling, it is recommended to use a specialized punch or scalpel for sampling and to prevent cross-contamination by cleaning the punch or scalpel with 2% sodium hypochlorite solution between each time of sampling. To amplify DNA with high GC content, DMSO can be added to PCR reactions at a final concentration of 1-10% (v/v).Note 2: Each Primer at a final concentration of 0.5 µM normally works well, but primer concentration can be optimized between 0.1-1.0 µM. Increase the primer concentration when amplification efficiency is low and decrease the primer concentration when non-specific PCR products are amplified. a. Mix well by gentle vortex or pipetting. Centrifuge briefly to allow liquid to accumulate at the bottom of the PCR tube.b. (Optional) When using a thermocycler without a hot lid, add a drop of mineral oil to the reaction to avoid evaporation. 2. Transfer the PCR reactions to a thermal cycler and run thermocycling conditions as follows:StepTemperatureDurationCyclesInitial Denaturation94℃5 min1Denaturation94℃30 sec30-40Annealing55℃30 secExtension68℃2 min/kbFinal Extension68℃10 min1Holding4℃--Note 1: Denaturation at 94℃for 5 min is to lysis plant tissues to release genomic DNA for PCR amplification.Note 2: Optimize PCR running conditions based on the template, primer sequence, amplicon length or GC content, etc. Note 3: Use 35 cycles for your first try to ensure obtaining the expected DNA fragments.3. Analyze PCR products by agarose gel electrophoresis: After amplification, centrifuge PCR reactions at 3000-5000×g for 3-5 minutes to collect the supernatant for agarose gel electrophoresis. This product contains premixed loading dye that enables reactions to be loaded directly on the agarose gel.FAQ:1. No product at all or low yield.a. Primer sequence is not well designed. Use primer design tools to avoid inappropriate GC content, secondary structure, dimer, annealing temperature, length, specificity and other possible problems. When adding restriction enzyme cutting sites in the primer sequence, the same problems need to be considered. In the case that positive control primers work well but not your primers, redesign primers.b. DNA may have a high GC content. High GC genes are relatively difficult to be amplified. In this case, GC-Rich buffer suitable for amplifying GC-rich DNA fragments is recommended, and PCR reaction parameters should be adjusted accordingly.c. DNA fragment is too long to be amplified. Although PlantTaq™ DNA polymerase contained in the Master Mix can amplify DNA fragments up to 5 kb in length, it is more suitable for amplification of DNA fragments less than 2-3 kb. Optimize primer sequence or other PCR parameters to amplify longer DNA fragments. d. The presence of secondary structure in primers, primer dimmers or short primers, may result in poor annealing of primers to the target sequence. Try touch-down or other methods for annealing. A gradual cooling from 65ºC to 55ºC or 50ºC can usually make annealing more efficient.e. Annealing temperature needs to be optimized. If necessary, use a temperature gradient to find the optimal annealing temperature for each template-primer pair combination. f.Insufficient extension time. Use an extension time of two minutes per 1000 base pairs. For DNA fragments hard to be amplified, try 3-4 min per kb. g. Possible problems of PCR thermal cycler. Use a different thermal cycler. h. PCR cycle number is insufficient. Try more PCR cycles. Not to exceed 40 cycles in general.i. The amount of DNA template is too low. Add more plant tissues but within the tolerant limit of the Master Mix, or try nested PCR or secondary PCR. Nested PCR is to design a pair of PCR primers inside the originally designed PCR primers, and then conduct PCR amplification again with the diluted first PCR product as a template. Instead, secondary PCR uses the same primers for PCR amplification again with the diluted first PCR product as a template. Nested PCR can usually remove the non-specific DNA amplification, but secondary PCR can not. j. Use desalted, PAGE or HPLC-grade PCR primers. k. When non-specific DNA fragments are amplified, increase the annealing temperature appropriately.l. Positive control and negative control are always recommended when optimizing PCR reactions. 2. Presence of non-specific PCR products or DNA smear.a. Increase the annealing temperature by 2-5℃.b. Reduce the amount of plant tissues in PCR reactions.c. Assemble the PCR reactions on ice. Non-specific products are produced easily if PCR reactions are set up at room temperature.d. 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