How can I improve PCR and get better access to the end band?

2017-08-28 21:38:46 GMT+0800

What is the system of PCR?

What about the process of PCR?

Within 1min, it's not possible that all of your friends will be able to say goodbye to the biological world.

The system is nothing more than a template, DNA polymerase, dNTP, primer, buffer, Mg2 + and water, and the reaction process of PCR is the legendary degeneration, annealing and extension.

Okay, so what are we thinking about when we can't add to the end strip, and when we're empty?

Let's open the door to science with a serious and serious attitude.

1.template

After all, the success of the PCR experiment is the template.

If the source of the experiment is wrong, how can the experiment be successful?

Some templates are not properly preserved, and when they are placed too long or have been degraded, the template needs to be replaced.

Some template extraction operation skill is not strong, resulting in the template is not pure, containing taq polymerase inhibitors or miscellaneous protein, at this time should be purified template and to extract, and detect whether the template contains inhibitors.

In addition, the concentration of the template is too high or too low to affect the results of PCR.

PCR sensitivity is high, or plasmid template with PCR products, only need around 1 ng, if use genomic DNA, or cDNA template, template amount can be appropriately increased, on the concentration gradient prediction can be based on the template of the experiment, so as to determine the appropriate concentrations of the template.

How can I improve PCR and get better access to the end band?

2.primers

The specificity of primers is very important.

Primers in the conservative area of the template design, best length of around 20 bp, GC content should be less than 60%, the 3 'end cannot have more than three consecutive G or C, to avoid primer itself to form dimers.

Design is completed, can undertake the BLAST primer, detect the specificity of the primer, or directly with NCBI prime BLAST primer design, some primers degradation extremely easily, so must pay attention to the operation and kept at low temperature on the ice.

3.reagents

Ensure that DNA polymerase activity did not even have the deactivation, the reagent, every time. This requires attention to timely back into the fridge, of course, to be able to save a few months at room temperature the BenchTop Taq mix is an exception.

4.reaction conditions

The first is the Tm value, and the importance of Tm in the PCR process does not need to be explained.

The Tm value is too high, and the primer is difficult to combine with the template.

Tm values are too low to be specific and prone to stray belts.

Generally speaking, the Tm value = 4 (G + C) + 2 (A + T), and the PCR set when renaturation temperature value = Tm - (5 ~ 10 ℃), among them A, T, G, C, respectively the number of corresponding bases.

20 base primer, for instance, if (G + C) % content was 50%, the operating temperature can be set to 55 ℃.

But if you think that addition and subtraction wasted your experiment for scientific research devoted to the good times, your order primers company will with the primer with primer purchase order, with your name when ordering the sequence of primers and Tm values.

Value - but it is important to note that Tm (5 ~ 10 ℃) is a experience value, if still cannot successful amplification (because of some primers working temperature will be greater than the Tm value), please be sure to!

Set the temperature gradient!

After getting the most comfortable temperature, then diving into the ocean of science...

Second, when setting up the PCR program, make sure to set the appropriate extension time.

General taq polymerase is an extension of time 1 KB/min, according to the extension of the length of the amplification products set up enough time, if the extension of time is not enough, DNA polymerase and dedicated on template amplification, temperature jumped suddenly, double-stranded DNA open, began to degeneration...

It leads to an increase in the number of hybrids, which you think is a hybrid, which is actually an incomplete purpose band.

5.reaction system

The reaction system can be optimized.

For example, Mg2 +, Mg2 + affects multiple aspects of PCR, such as DNA polymerase activity.

The optimal concentration of magnesium ions is different for different primer pairs and templates. In most cases, higher free magnesium concentration can increase production, but also increase nonspecific amplification.

In order to determine the optimum concentration, the Mg2 + with increasing concentration of 0.1-5mmol/L can be used to select the optimal Mg2 + concentration.

In the PCR reaction mixture, it is necessary to minimize the concentration of negatively charged groups, such as phosphate groups or EDTA, which may affect Mg2 + ion concentration to ensure optimal Mg2 + concentration.

In addition, there are legendary substances called PCR additives, including formamide, DMSO, glycerin and betaine, which are commonly used in DMSO and glycerin.

Their mechanism is to reduce the melting temperature, thereby helping to induce annealing and to assist the DNA polymerase to extend through the secondary structure.

The effect of glycerin and DMSO is the same: reduce the combination of the double chain and reduce the non-specific generation.

However, different PCR reaction systems, additives play a different role.

So, if you use PCR additives, you need to optimize the concentration gradient.

Added too little, the additive does not play an effective role.

Adding too much, the additive can affect and even inhibit PCR amplification.

Glycerine and DMSO do not need to be added at the same time, add 5% glycerin or 10% DMSO.

If the concentration is still not work, you can choose to set up the gradient, after all the different experimental conditions corresponding to different experimental system, the experiment is not fixed, it is often necessary to us according to particular case is particular analysis, to get a satisfactory result.