Questions about primers for PCR cloned genes

You see, you get 1-1200 nt and 1000-1400 nt sequences, respectively, so the 200 bp sequences in the middle are overlapping, right? That is, you have obtained all the sequence information in this CD area from the sequence information. If you just need sequence information, this is sufficient, but if you need to amplify the full 1400 bp for subsequent experiments, then you need to continue to amplify the full CD region.

If that's the case, I have a trick, you can mix and use the 1-1200 nt and 1000-1400 nt fragments as templates, and then use primers A and D for amplification, you can easily get a complete 1400 bp product, but the premise is that when you denature annealing in the first step, the time is appropriately longer, so that the two large fragments can be mixed and annealed, that is, the 200 bp part of the two fragments overlapping can be annealed, which is convenient for you to amplify.

PCR reaction conditions. When amplifying with primers A and D, is the extension time of the 72-degree extension step sufficient?

If you need the complete CD region for subsequent experiments, then you need to continue to amplify the CD region intact.

You can do a gradient PCR, and the effect is obvious; Alternatively, you can use LA Taq. Hope it helps

One primer is complementary to one strand of DNA template at one end of the target region.

The primer is a synthetic two-segment oligogroup silver tolerant nucleotide sequence, one primer is complementary to a DNA template strand at one end of the target region, and the other primer is complementary to another DNA template strand at the other end of the target region, its function is to serve as the starting point of nucleotide polymerization, and nucleic acid polymerase can synthesize a new nucleic acid strand from its 3 ends.

The purpose of PCR primer design is to find a suitable pair of nucleotide fragments that allow them to efficiently amplify template DNA sequences. As mentioned earlier, the quality of primers is directly related to the specificity and success of PCR. There cannot be an all-encompassing rule for the design of primers to ensure the success of PCR, but following certain principles can help in the design of primers.

PCR primer design principles.

1. The length of primers is generally 15-30bp.

The length of primers is generally 15-30 bp, and the commonly used one is 18-27 bp, but it should not be greater than 38 bp, because too long will cause its elongation temperature to be greater than 74, which is not suitable for Taq DNA polymerase reaction;

2. The GC content of primers is generally 40%-60%.

The GC content of primers is generally 40%-60%, and 45-55% is appropriate, and too high or too low GC content is not conducive to initiating the reaction. The GC content and TM value of upstream and downstream primers should be kept close;

3. The TM value of the template sequence corresponding to the primer should be about 72.

A tm value of around 72 can make the refolding conditions optimal, at least between 55-80. 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;

4. The base at the 3' end of the primer generally does not use A.

The base at the 3' end of the primer generally does not use A, because A is relatively efficient at the wrong priming site.

5. The presence of more than 3 consecutive bases at the 3' end of the primer, such as GGG or CCC, will also increase the probability of error.

6. The complementary, dimer or hairpin structure at the 3' end of the primer may also cause the PCR reaction to fail.

7. If the coding region is amplified, the 3' end of the primer should not terminate at the 3rd position of the codon, because the 3rd position of the codon is prone to degeneration, which will affect the specificity and efficiency of amplification

8. The 5' end of the primer can be modified.

The 5' end sequence of primers has little effect on PCR, so it is often used to introduce modification sites or markers.

9. The bases should be randomly distributed, and there should be no 4 consecutive bases of complementarity between the primer itself and the primer.

The role of primers in PCR: to find a suitable pair of nucleotide fragments that allow them to efficiently amplify the template DNA sequence.

Primers are artificially synthesized two-segment oligonucleotide sequences, one primer that complements one strand of DNA template at one end of the gene of interest, and the other primer that complements another strand of DNA template at the other end of the gene of interest.

In PCR (polymerase chain reaction) technology, the nucleotide sequence of a target gene is known, and primers are synthesized according to this sequence, and the DNA of the target gene is denatured by heat and denatured into a single strand, and the primers are combined with the corresponding complementary sequence of the single strand, and then extended under the action of DNA polymerase, so that the cycle is repeated, and the product obtained after extension can also be combined with the primers.

The essence of the primer is the nucleotide sequence, which is used as the binding site of DNA polymerase at the beginning of DNA replication, and the specific PCR principle is to use DNA molecules to denature and unwind into a single strand at a high temperature of 95 in vitro, and then when it is cooled to about 60 °C, the primer will be combined with single-stranded DNA according to the principle of base complementarity, and then the temperature is raised to about 72 °C, that is, the optimal reaction temperature of DNA polymerase, and DNA polymerase from 3'The end begins to synthesize the corresponding complementary strand in the direction of the new nucleic acid strand, and so on to achieve the purpose of DNA molecule amplification.

can be found in primers.

Add unpaired bases, but at least three of the 3' ends of the primers are tightly paired. However, this operation is mainly for the purpose of adding enzyme cleavage sites or flags.

The tm value is calculated based on the annealing of the two strands, and the unpaired bases are not counted, so you add the unpaired bases to have little to no effect on the tm value.

Guaranteed length – there is no need to add unpaired bases.

No dimers are produced.

It is also not necessary, the base pairing of the primers itself has little effect on amplification, and the dimers between the primers can be avoided according to the choice of primer position, and if it cannot be avoided, it is necessary to ensure that there is no pairing at the 3' end.

Therefore, it is not recommended to add unnecessary bases to design primers. (Except for some interference experiments).

Not every base on the primer has to be complementary to the template strand, but the design like the one you see in the figure is not possible, generally the 5' end of the primer can be completely different from the template, so that you can have primer enzyme digestion sites and so on in the PCR, but the 3' end must be bound to the template strand, because the Taq enzyme is extended in the 5-3 direction, and if the 3' end is cocked, it cannot be extended. If you have a 3' end of the primer that binds to somewhere downstream of the template, it becomes a PCR of the deletion segment.

I don't think it's necessary, but the number of unpaired bases should be as small as possible, and key locations such as digestion sites or active sites should be as conservative as possible.

Primer 3'The terminal bases, especially the last and penultimate bases, should be strictly paired to avoid PCR failure due to terminal base mispairing.

In the PCR reaction, the new DNA strand is synthesized from the 3' end of the primer. Therefore, in the newly synthesized DNA strand, the original primer is at the 5' end, and the new reed mill adds the base at the 3' end.

The 3' end of the primer is the key base, which is the starting end of the PCR extension, and can not be modified in any way, nor can it form the possibility of secondary structure, and the 3' end can not be mismatched, and the pairing should be strictly required to avoid PCR failure due to the mismatching of the end bases.

The 5' end is not strictly restricted, and only plays the role of limiting the length of the PCR product, which has little effect on the specificity of amplification, so it is often used to introduce modification sites or markers. Accompany the fight.

Suitable digestion sites may be added to the primers, and the amplified target sequence should preferably have a suitable digestion site, which is very beneficial for digestion analysis or molecular cloning.

The concentration of each primer is 1umol or 10 100 pmol, which is the best result for the minimum amount of primer residue to be generated, as high primer concentrations can cause mismatches and non-specific amplification, and can increase the chance of dimer formation between primers.