Why can t the PCR run out, it s all dispersed

There are many reasons for diffusion:

1. Whether the PCR product is electrophoresis immediately after the end of amplification, and whether the electrophoresis solution is fresh, if it is not very likely to lead to degradation of the amplification product.

2. The PCR system has not been explored, use orthogonal experiments to find out the correct PCR system, don't do it simply based on other people's test data, don't believe too much in the data in the article, everyone will protect their own test results and data.

3. If the design of the primers used in amplification is not good, find someone else to help you see it, or find a biological company to help you design it. If it's a universal primer, make sure that the basics you want to amplify are highly conserved.

4. The problem of the template, whether the template you use is degraded. Is DNA the template, or RNA is the template.

Finally, if you start with multiple bands of amplification, it may be that the primer is not specific or the annealing temperature is too low. If you still have a completely diffuse phenomenon after exploring the conditions, it is recommended to change the primers.

Drag from head to tail, another pair of primers.

can make it."

Indicate that dimers are not formed between primers.

Rather, the design of the first pair of primers is problematic (specificity.

Very poor). If you must amplify that region, it is recommended that the primers be placed on either side. If you don't have good design software, it is recommended to try primer3 (the best free primer design out there).

If that still doesn't work, send me the sequence and I'll design it for you (I have professional software, ABI Primer Express 3 and Invitrogen Vector NTI 10).

It is usually caused by excessive non-specific amplification, which is raisedAnnealing temperatureTry it.

If the PCR product is left for too long, the product degradation should be considered; If the sample is loaded in time after PCR, consider primersSpecificityPoor or impure templates (including degradation and adulteration of other DNA sequences).

If the PCR product is used as a template, the amount is too large.

The glue is not cooked well, and it will be the same.

Stripe is a method of splitting contiguous data into blocks of the same size, and writing each piece of data to different disks in the array. To put it simply, striping is a method of merging multiple disk drives into a single volume. In many cases, this is done through a hardware controller.

Disk conflicts may occur when multiple processes access a disk at the same time. Most disk systems have limits on the number of accesses (IO operations per second, IOPS) and data transfer rate (the amount of data transferred per second, tps). When these limits are reached, the processes that need to access the disk will have to wait.

This is known as a disk conflict. Avoiding disk conflicts is an important part of optimizing IO performance, and optimizing IO performance is very different from optimizing other resources (such as CPU and memory), and the most effective way to optimize IO is to balance it to the maximum.

Striping is a form of automatic load balancing of iO.

Striping is the technology that divides a piece of contiguous data into many small parts and stores them on different disks. This allows multiple processes to access multiple different parts of the data at the same time without causing disk conflicts.

And when you need to access this data sequentially, you get the maximum amount of IO parallelism, which results in very good performance. Due to the superior performance of striping in terms of IO performance, striping technology is involved at multiple layers or platforms in the computing environment where the application system is located, such as the operating system and the storage system.

First of all, suspicion, specificity is not good, solution:

1. It may be that the amount of template is too high, and the concentration of template is reduced by 10-1000 times;

2. Appropriately increase the annealing temperature by 1-3 degrees, or use 1-3 degrees higher than your previous annealing temperature for 5 cycles, and then use the remaining cycles of the previous temperature p (that is, set 2 more steps in the program);

3. Slightly reduce the concentration of mg ions, if you have added it before, try it;

If the above does not resolve the issue, check your template, primers, and Taq, re-remove the template, re-dilute the primers, and use a new PCR reagent.

If you have any questions, please continue to discuss, it is purely hand-made, welcome to adopt, I wish the experiment a smooth ride!

The problem with the Taq enzyme is the most likely, and it is also possible that the primers have been degraded by contact with the RNase.

It's best to change everything you want to add to your PCR system, and if it was done well before, the template should be fine.

I also used cDNA as a template, and there were no diffusion bands, but non-specific bands often appeared. The cause of the diffusion band may be that the electrophoresis solution is contaminated or there is a problem with the gel you are doing, or it may be that your cDNA is partially degraded, and the reason for the degradation is mainly due to time and temperature. After amplification, electrophoresis should be carried out immediately, and secondly, the room temperature of electrophoresis should not be too high, and the time should not be too long, generally about half an hour at most.

In addition, in the process of amplification, due to the climate, some PCR instruments can not work normally after the room temperature exceeds 30 degrees, which depends on the model of the PCR instrument, followed by the poor effect of RNA extraction in the early stage, or the quality of the cDNA template has been affected due to the degradation of the extracted RNA.

Finally, about the PCR part, the system and annealing temperature of PCR are very important, especially the concentration of magnesium ions, it is best to use orthogonal tests to find out the best system, and then use the temperature gradient to find the most suitable annealing temperature. Because the quality of the design is also one of the major problems, hehe! Let's try it first!

There are several reasons for this.

1. The problem with PCR itself is not that it is a PCR machine. Maybe the primers are not well designed, or it may be that the return temperature is not the most suitable.

2. The DNA is too thick. Generally, the total amount of DNA should not exceed 100 ng for a 25 microliter system.

3. The gel is not ready. Generally, it is ready to use, and it should not be left outside for more than 2 hours.

The first one is the most likely.

If there is a band dispersion, it may be that the amount of template is large, or the conditions are not suitable, such as temperature, time, cycle number or something; If there is no band diffusion, it is generally that the PCR response is not successful.

Lilac Garden and Little Wood Worm have a lot of posts and experts in this area.