Principles and experimental methods of PCR technology (3)

Three, case analysis-black snake

(I.

In the process of processing, the main distinguishing characteristics of the black-snake pieces have almost disappeared, and it is difficult to accurately identify them through appearance characteristics.

(2) Setting of temperature and time

Based on the three-step PCR principle, three temperature points of denaturation-annealing-extension are set

(3) Principles of primer design

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Avoid secondary structure and hairpin structure in primers, and avoid complementation between two primers, especially the complementation at the 3'end, otherwise primer dimers will be formed and non-specific amplified bands will be formed;

It is best to use G or C at the 3'end of the primer, but do not GC, especially the last and penultimate bases.

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(4) Analysis of common problems and countermeasures

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(5) Reagents needed

Animal genome extraction kit, liquid nitrogen, high-fidelity TaqDNA polymerase and buffer, dNTP Mixture, DNA molecular weight marker, nucleic acid gel stain GelRed, agarose, identification primer (synthesis)

The various molecular biology techniques RAPD, AFLP, ISSR, PCR-RFLP, AR-PCR, SCAR, SRAP, TRAP, ARMS, DNAbarcoding, etc.

Related Links: Principles and Experimental Methods of PCR Technology (2)