Q question: Are the normal PCR and QPCR primers the same?
A answer: Â The design principles of the two are the same and different;
The same place:
•  The search for sequences is consistent;
•  Sequence selection should be in a conserved segment of the gene;
•  Select the appropriate size of the amplified fragment
•  Avoid forming 4 or more consecutive pairs between the primer itself or with the primer;
•  Avoiding the primer itself to form a ring-shaped hairpin structure;
•  Tm value is 55-65 ° C, GC content is 40%-60%;
•  The TM phase difference between the primers is avoided to exceed 2 ° C;
•  Avoid 3 or more consecutive identical bases at the 3' end of the primer;
Different places:
Real time PCR Â Primer
•  PCR product length; real-time PCR is required to be within 300 bp, generally preferred between 80-150 bp;
•  Multiple pairs of target genes are simultaneously amplified, and the primer conditions found in the literature will be different, and it is necessary to design primers that are as consistent 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; the melting curve requires a single product;
•  The purpose of Real time PCR is to perform quantitative or relative quantification, which requires the efficiency of amplification; high requirements on the secondary structure of the primer;
Common PCR primers:
•  According to the requirements of the experiment, the length generally ranges from 150bp to several thousand bp;
•  There is no real time PCR high for secondary structure requirements;
•  Low requirements for amplification efficiency;
•  Gradient PCR instrument can be used to select different annealing temperatures, and the annealing temperature requirements of the primers need not be consistent;
Different when verifying:
Primer specificity (same point):
Ø  The specificity of the primer is guaranteed by blast before use;
Difference: real time PCR
Ø  Verification by melting curve in the experimental results;
Ø  The specific product peak of the 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, running the strip;
Real time PCR can identify the relative amount of product through amplification curve and melting curve analysis. It can also perform electrophoretic analysis on the final product, which is more comprehensive and scientific!
Ground-Inserting Micro-Spray,Rod-Inserting Micro-Spray,Fruit Tree Vortex Micro Nozzle,Greenhouse Hanging Micro Spray
Shandong Yibiyuan Water-saving Equipment Technology Co., Ltd. , https://www.cnybyjs.com