1. If the ct value of the amplification curve is lower, 32 or so, what is the general solution?
The ct value is relatively low, which has a certain effect on the experiment, because after so many cycles, the activity of the enzyme may decrease, and the amplification efficiency will change at this time (the theoretical basis of quantitative PCR is each cycle). The amplification efficiency we consider is a certain value). Therefore, it is best to move the ct value forward.
Solution The template can be concentrated or drained and dissolved with a small amount of water.
2. Why does the synthetic primers amplify the target band without the comparison of the template, and the internal reference does not have the same system?
The primers are contaminated.
3. Real-time quantitative PCR, real-time PCR, real-time PCR, these are the same concept
Is a concept.
4. The slope of my standard curve is always greater than 4, and the parallel ct value of each gradient is quite different. Why?
This slope is = 1/LOG 10 (amplification efficiency), theoretically the amplification efficiency = 2, and the slope is 3.322. If the slope is greater than 4, the amplification efficiency is relatively small. Can you check if the reaction system is reasonable? Is the enzyme activity normal?
Parallel tubes have large differences in CT values. You can check whether your experimental operation is standardized. Another reason is that the error of the instrument itself may cause a large difference in CT values.
When the slope is 4, the amplification efficiency is 77.8%, and if the slope is greater than 4, the efficiency continues to decrease.
The problem of amplification efficiency is actually the amplification efficiency of the primers, and the relationship between the enzyme activities may not be so important, provided that the quality of the kit is guaranteed. Returning to the amplification efficiency, the satisfactory amplification efficiency can be obtained only when the primer and the template are at the optimum ratio. Therefore, by adjusting the final concentration of the primer in the PCR reaction system, some primer gradients can be compared for comparison.
5. What is the role of ROX dyes?
The role of ROX ABI claims to be used to calibrate the loading error.
But it is said that the role of ROX is actually used to calibrate the optical path difference. That is, the fluorescence signal of each hole through the filter is not different when the focus is CCD, so the brightness of the image on the CCD is different. So you need to use ROX to calculate how much the difference between the holes is, and then the difference coefficient to deal with the actual fluorescent signal. I am not very clear about the specific process, please be expert.
6. How is the baseline of real-time PCR determined?
The baseline is the background value, so it is the segment of the curve before it has "taken off". There is a place on the software to enter the baseline from the first few to the first cycle as a baseline. The setting principle is to make the signal of the most cycle before the jump is close to zero.
7. I am just getting ready to do the quantification. I would like to ask, how about the machine of Bole? Please recommend a few suitable SYBR GREEN boxes, less than 1000 (no money is not easy to do), I will do about 20 samples. Is it approved for SYBR GREEN now?
Bole's machine is good. Takara's kit, about 1,500 yuan, 400 reactions. Most people still use sybr green I. This is the most commonly used dye.
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