Products
This product is a high purity genomic DNA extract from 293T cells, agarose gel (0.7%) electrophoresis showed that the size of the DNA extract is more than 15Kb, and basically no degradation, the product is ultimately preserved in TE Buffer, which can be widely used in molecular biology experiments, such as PCR, enzyme digestion, hybridization, microarray analysis, and other molecular biology experiments.
The product was quantified using NanoDrop One at a concentration of 200 ng/μL.
Preparation and precautions before use
Long-term storage at -20˚C is recommended. Before use, the bottle should be removed from the refrigerator and equilibrated to room temperature and centrifuged before opening the cap for use. Samples should be restored to the sealed state as soon as possible after opening.
How to use (take qPCR experiment as an example)
1. Amplification template preparation
The samples to be detected were diluted with TE (10 mM Tris-Cl, pH 8.0,1 mM EDTA), and the concentration after dilution was as close as possible to the range of 0.05-10 ng/μL. The samples were placed on ice at 4°C and set aside.
2. Standard dilution: according to the following table, firstly dilute Human DNA Standard 1 (100ng/uL) with TE to make 5 different concentrations of standards according to the table below. 10ng/μL of DNA Standard 1 (Std. 1) can be stored stably at -20℃ for 1 month; Std2-5 can only be used on the same day, and should be placed at 4℃ or on ice when not in use for the time being after preparation. When not used temporarily after preparation, it should be stored at 4℃ or on ice.
style | Corresponding concentration (ng/μL) | Minimum dilution volume (in μL) | Std.1 | 10 | 10 [100 ng/μL DNA Standard 1] + 90 TE | Std.2 | 2.5 | 20 [Std. 1] +60 TE | Std.3 | 0.625 | 20 [Std. 2] +60 TE | Std.4 | 0.15625 | 20 [Std. 3] +60 TE | Std.5 | 0.0390625 | 20 [Std. 4] +60 TE |
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3. qPCR reaction system preparation
The cryopreserved reagents to be used were completely thawed and mixed by inversion several times before preparation, and then briefly centrifuged and prepared for use. 20 μL of the base reaction system was as follows.
The base reaction system for 20 μL was as follows:
reagents | 20μL reaction system | 2×qPCRMix | 10μL | PrimerMix | XμL | ProbeMix | XμL | Template | 4μL | ddH2O | Make up to 20 μL |
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Note: High Rox model: add 1 μL of 50×High Rox per 50 μL of reaction system; Low Rox model: add 1 μL of 50×High Rox per 500 μL of reaction system.
Usually, better results can be obtained with a primer concentration of 0.2 μM, and 0.1-1.0 μM can be used as a reference for setting the range.
The concentration of the probe used is related to the fluorescent quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance, so please refer to the manual of the instrument or the specific requirements for the use of each fluorescent probe for the adjustment of the concentration during actual use.
Prepare a sufficient amount of reaction system mixture as required. After the reaction system has been prepared and mixed thoroughly, add 16 μL per well to the reaction wells. Then add the prepared standard and diluted sample into the corresponding reaction wells, the volume of addition is 4μL/well. TE was added to the blank control tube, and the same amount of TE was added at 4 μL/well.
It is recommended to use 20 μL for the reaction, if you need to perform a smaller system reaction, reduce the system components in equal proportion.
4. qPCR reaction program
The following is an example of our GoldStar Probe Mixture reaction conditions, which should be improved and optimized according to the PCR product template, primer structure and target fragment size.
move | temp | timing | circulate | premutability | 95°C | 10min | 1 | denaturation | 95°C | 10sec | 55 | Annealing/Extension | 60°C | 30sec | 5 |
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Data analysis
1. Standard curve production
The standard curve was plotted with reference to the Excel sheet for data processing. The correlation coefficient R2 of the standard curve should not be lower than 0.98, and the slope should be between -3.1 and -3.6 when the Ct value is the vertical coordinate. If the parameters of the standard curve are unreasonable, it is recommended to repeat the experiment.
