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Common PCR and QPCR primers similarities and differences

Views:1     Author:Site Editor     Publish Time: 2022-01-13      Origin:Site

The same:

• The search of the sequence is consistent;

• The sequence selection should be in the conserved segment of the gene;

• Choose the appropriate amplified fragment size

• Avoid the formation of 4 or more consecutive pairs of primers themselves or with primers;

• Avoid the primer itself forming a circular hairpin structure;

• Tm value is 55-65℃, GC content is 40%-60%;

• The TM difference between primers should not exceed 2°C;

• Avoid 3 or more consecutive identical bases at the 3' end of the primer;

Difference:

Real time PCR primers

• Length of PCR product; real-time PCR requires within 300bp, generally 80-150bp is preferred;

• Multiple pairs of target genes are amplified at the same time, and the primer conditions found in the literature will be different. It is necessary to design primers with the same conditions as possible;

• When the target gene content is relatively low, it is necessary to design primers with relatively high sensitivity;

• Relative to electrophoresis, Real time PCR has higher sensitivity, higher requirements for primers, and fewer primer dimers; melting curve requires a single product;

• The purpose of Real time PCR is to perform quantitative or relative quantification, which has requirements for the efficiency of amplification; high requirements for the secondary structure of primers;

real time pcr

Common PCR primers:

• Depending on the requirements of the experiment, the length is generally from 150bp to several thousand bp;

• The requirement for secondary structure is not as high as that of real time PCR;

• The requirement for amplification efficiency is not high;

• Gradient PCR instrument can be used, and different annealing temperatures can be selected, and the requirements for primer annealing temperature do not need to be consistent;

Different when verifying:

Primer specificity (same point):

Ø The specificity of primers is guaranteed by blast before use;

Difference: real time PCR

Ø Verified by the melting curve in the experimental results;

Ø The specific product peak of PCR should be within two degrees of the PCR product in the primer report;

Ordinary PCR identifies the specificity of the product by the length of the product and running the band;

Real time PCR can identify the relative amount of products through amplification curve and melting curve analysis, and can also perform electrophoresis analysis on the final product, which is more comprehensive and scientific!


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