PCR a gene of more than 2000 bp primer design

The length of the target product is generally of little relevance to the primer design. When designing, the following points should be noted:

1.The length of primers is generally 15-30 bp, 18-27 bp is commonly used, but should not be greater than 38, as too long will cause its elongation temperature to be greater than 74, which is not suitable for Taq DNA polymerase reactions[2].

2.The primer sequences should not have high similarity within the template, especially the sequences with high similarity at the 3' end, otherwise it is easy to cause mismatch. The presence of more than three consecutive bases at the 3' end of the primer, such as GGG or CCC, also increases the probability of false initiation [2].

3.The last base at the 3' end of the primer has a great influence on the DNA synthesis efficiency of Taqase. Different last bases lead to different amplification efficiencies at the mismatch position, and the mismatch efficiency of the last base A is significantly higher than that of the other 3 bases, so the use of base A at the 3' end of the primer should be avoided [3][4].

In addition, primer-dimers or hairpin structures can cause PCR reaction failures. The 5' end sequence has little influence on PCR and is therefore commonly used to introduce modification sites or markers [2].

4.The GC content of the primer sequence is generally 40-60%, and too high or too low is not conducive to initiating the reaction. The GC content of the upstream and downstream primers should not differ too much[2][5].

5.A TM value of around 72 for the template position sequence corresponding to the primer makes the refolding conditions optimal. There are several ways to calculate the TM value, such as according to the formula TM 4(G+C) 2(A+T), and the nearest neighbor method is used in the Oligo software [6][7].

6.The δg value refers to the free energy required for the formation of DNA duplexes, which reflects the relative stability of base pairs within the duplex structure. Primers with a low ΔG value at the 3' end (no more than 9 absolute values) and a relatively high ΔG value at the 5' end and intermediate should be selected.

The δg value of the 3' end of the primer is too high, which makes it easy to form a double-stranded structure at the mismatch site and initiate a DNA polymerization reaction [6].

7.The energy value of primer-dimers and hairpin structures is too high (exceeding them can easily lead to the formation of primer-dimer bands and reduce the effective concentration of primers, which prevents the PCR reaction from proceeding normally [8].

8.Primer modifications are generally done by adding an enzyme cleavage site at the 5' end, which should be determined according to the corresponding sequence of the vector into which the PCR product will be inserted in the next experiment.

It is worth mentioning that the difficulty of primer design varies from template to form. Some of the templates themselves are more difficult, such as high or low GC content, resulting in the inability to find primers that are suitable for various indicators; In PCR used for cloning purposes, the product sequence is relatively fixed, and the degree of freedom of choice of primer design is low. In this case, we can only settle for the next best thing and try to meet the conditions.

There are a lot of rules for PCR primer design on the Internet, and I believe you have probably read it.

Take it easy, the pp5 I used at the time is not to say that you can't get p out if the primer score of the primer he designed is low, these are not absolute. The first time you design primers, it's not so calm. In fact, you can ask your brothers and sisters to teach you so that you don't delay your experiment.

You can also try to design a pair or two of primers and try it out with PCR. Because PCR is not so difficult after all, of course, I didn't understand your topic, if you really want the product of PCR to have more than 2000 bp, it is also a bit troublesome. Like when I used to do prokaryotic expression, the basic primers were fixed at both ends, and they could still be p-out.

Recently, a classmate of mine did PCR, and the product is also 2000BP, and he just solved this problem, because of the problem of mg ion concentration, it is also important to optimize PCR conditions. In fact, generally speaking, according to a general system, this is done, the buffers of Takara are ready-made, and the mg ions are separated.

In fact, after talking for a long time, I didn't design the primers for you. I just want to say, take it easy ... Too many rules and regulations are useless.,My classmates they design primers and just look at them with your eyes.,Do you have to score 100 systematically?,Of course, considering the cost of the experiment.,Try it.,good luck。。。

There is no need to go through the hassle, just select your own amplified fragment and select a sequence of 18-25 bp from both ends as primers.

It's okay, as long as you don't form a typical loop, or the two primers are seriously paired (less than 50% is fine) The most important thing is that it can be amplified, and it's useless to think about it so much, after all, the experiment is tried.

Besides, synthetic primers are not expensive now

Summary. Hello classmates!

A primer is required for the synthesis of each daughter strand. PCR replicates n times, the number of DNA produced is 2 N, each DNA has two strands, a total of 2 N*2=2 (N+1), and then subtract the two parent strands, so at least the N+1 power of the number of primers 2 needs to be added minus 2.

A pair of primers is required for each replication. In general, the primers added to the reaction are excessive.

A "primer" is a small piece of DNA that serves as a starting point for DNA replication, and when one replication is completed, the primer is not on a strand, and the next replication is done, the primer also needs to be replicated.

How many of the DNA containing one of the primers were obtained in PCR amplification.

Hello classmates! The synthesis of each sub-chain requires a lead. PCR replicates n times, the number of DNA produced is 2 N, each DNA has two strands, a total of 2 N*2=2 (N+1), and then subtract the two parent strands, so at least the N+1 power of the number of primers 2 needs to be added minus 2.

Each copy requires a pair of primers. In general, the primers added to the reaction are excessive. A "primer" is a small piece of DNA that acts for:

As the starting point for DNA replication, when one replication is completed, the primer is not on the same strand, and the next time the replication is made, the primer also needs to be replicated.

How many primers does the replicated DNA contain?

Primers are required for DNA replication, and if a DNA is replicated n times, at least 2N+1-2 primers need to be added to the buffer. For each replication of a DNA molecule, a pair of primers is required from the jujube family, replicated n times, a total of 2n DNA molecules, 2n+1 primers are needed, and a DNA molecule of the parent is needed (no need for primers from Xiaoyan), so it is necessary to add at least 2N+1-2 primers to the buffer.

Why doesn't one of the parent's DNA molecules need primers?

This is due to the difference in the nature of "DNA polymerase" and "RNA polymerase". DNA polymerase, which is resistant to action, must be preceded by a small piece of RNA primer; RNA polymerase, which does not require any primers, can be synthesized from scratch. DNA synthesis requires primers, which can ensure the high fidelity of DNA replication, because the synthesis of primers is very unstable and easy to enter the wrong base, so after DNA synthesis, the primers will be excised, and then supplemented with DNA to make it complete.

DNA is genetic material, so there is this error-proofing replication system. RNA is less important, so it does not have a sophisticated error-proofing system like Muqing.

Will DNA contain two primers?

Yes. A gene was amplified four times, and the resulting DNA contained a total of 15 of the primers. Why.

If it is a double strand, the first cycle should contain 1 pair of primers, the second beam bond should be 2 pairs, the third oak rock qiao should be 4 pairs, and the fourth should be 8 pairs. A total of 15 pairs. There were 15 upstream and downstream primers.

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The role of primers in PCR is to act as a binding site for DNA polymerase at the beginning of DNA replication, and under extracellular conditions, DNA can only begin to replicate through primers.

A synthetic two-segment oligonucleotide sequence, with one primer complementing one strand of DNA template at one end of the region of interest and the other primer complementing another strand of DNA template at the other end of the region of interest.

There are two primers in the PCR reaction, the 5-terminal primer and the 3-primer. Primers are designed based on a single strand of DNA (often based on the information strand), and the 5-end primer is the same as a small piece of DNA on the 5-end of the fragment to be amplified; The 3-end primer is complementary to a small piece of DNA sequence located at the 3rd end of the fragment to be amplified.