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Products & Features:
Developed by our companyEnterobacter is a universal dye PCR kit, which has the following characteristics:
1.Out-of-the-box, the user only needs to provide a virus sample.
2.Specific primers designed according to the generic conserved sequence of Enterobacter spp., with no cross-reactivity with related viruses.
3.Sensitivity can reach hundreds of copies of the reaction.
4.One-tube PCR detection to avoid subsequent contamination.
5.This kit is sufficient for 50 20 L reactions.
Transport & Storage:Shipped at low temperatures, -20 stored, valid for one year.
Bring your own reagents:DNA template, 10 ROX (depending on the model, see how to use).
How to use 1Dilute the PCR positive control (take the 6 10-fold dilutions of 10E2-10E7 copies L as an example).
1.Note: Due to the very high concentration of the standards, the following dilutions must be performed in a separate area and must not contaminate the sample or other components of this kit). In order to increase the stability of the product and avoid the spread of infectious pathogens, this product does not provide live samples as a positive control, only DNA fragments that can be used directly as a positive control.
2.Label 6 centrifuge tubes, 7, 6, 5, 4, 3, 2.
3.Add 45 L of fluorescent PCR-specific template dilution with a core tip (preferably with a core tip, the same below).
4.Add 5 l of 1 10e8 copies of the positive control to tube 7 for 1 min to obtain a positive control of 1 10e7 copies. Set aside on ice.
5.Change the pipette tip, add 5 l of 1 10e7 copy of L of positive control (diluted in the previous step) to tube 6, and fully ** 1 min to obtain 1 10E6 copy of L of positive control. Set aside on ice.
6.Change the pipette tip and add 5 L of 1 10E6 copy of the positive control to the No. 5 tube for 1 min to give 1 10E5 copy of the positive control. Set aside on ice.
7.Repeat above until you get a positive control with 6 dilutions. Set aside on ice.
2. Preparation of sample DNA:
8.If there are n samples, n+2 extractions must be set, with the extra one being PC (Sample Preparation Positive Control) and one being NC (Sample Preparation Negative Control). PC can be used as PC with 10 L of PCR positive control No. 4 (1 10e4 copies L, 10 L is equivalent to 10,000 copies) or No. 5 (1 10E5 copies L, 10 L is equivalent to 100,000 copies) plus a certain amount of water to make the total volume the same as the volume required for each preparation. In addition, water is used as NC. If 200 L of sample is required per preparation, the volume of PC and NC must also be 200 L.
9.Purify samples of DNA using a method of choice, and this kit is compatible with most viral DNA extraction kits on the market. The company's one-tube viral DNAout or its upgraded column viral DNAout can also be purchased as an option. This kit comes with 15 complimentary vibulal virals of viral DNAOUT.
3. Set up the qPCR reaction (20 L system, performed in the sample preparation room):
10.If only 1 replicate is made, label n+9 PCR tubes, with n+2 for the n+2 sample from the previous step, 1 for the negative PCR control, and 6 for the positive PCR control. If you do 2-3 replicates, increase the number of reaction settings by a factor of 2 or 3.
11.Add the ingredients to the labeling tube as shown in the table below (only one replicate is listed in this table.) Set up the positive control after the sample tube and negative control are set up, and the positive control sample should wait until all tubes are covered and stored.
Add later).
Note:ROX is required for the ABI and 7900 instruments only, and ROX is not required for other fluorescent PCR instruments such as the iCycler IQ, MJ Option, MJ Chromo4, MX3000, MX4000, Rotorgene3000, Rotorgene 6000, and Lightcycler480, and is replaced with water.
12.Close the lid and get on the machine, and perform PCR according to the following parameters (the specific PCR parameters can be optimized according to different instruments).
13.Data acquisition.
The operation is carried out according to the recommended process of the instrument used. The fluorochrome contained in this product has a maximum absorption spectrum of 471 nm for non-binding DNA, 500 nm for binding DNA, and 530 nm for binding DNA. Signal acquisition can be set up during the refolding or extension steps.
4. Data Processing:
14.If this kit is used for quantitative detection, the log value of the positive control concentration is used as the horizontal axis and the CT value is used as the vertical axis, and the standard curve is plotted. The CT value of the sample to be measured is then used to calculate the log value of the DNA concentration of the sample from the standard curve, and then its concentration is calculated.
15.If this kit is used for qualitative testing and only positive or negative is judged, the negative control CT must be greater than or equal to 40. The positive control must have a logarithmic increase in fluorescence, have a typical amplification curve, and the CT value should be less than or equal to 30. The sample to be tested is negative if its CT is greater than or equal to 40 and positive if it is less than or equal to 35. If it is between 35-40, repeat once. The CT value of the repeat experiment is negative if it is greater than or equal to 40 and positive if it is less than 40.
It is only used for scientific research, not for clinical diagnosis!All products are for scientific research use only and may not be used for other purposes.