Molecular cloning and plasmid vector construction experiment
Molecular Cloning and Plasmid Series Construction Experiment Molecular Cloning u0026 Nbsp; Service introduction
Molecular cloning is A method of purifying and amplifying a specific DNA fragment is provided at a molecular level. Often contains a target gene, inserting them into a cloned carrier, forming a recombinant cloning carrier, forming a suitable host, then introducing a suitable transfer host, and then separating purified by the selected host intracellular The needed cloning carrier can be obtained by inserting a copy of DNA to obtain an amplification of the purpose gene.Second,
Molecular cloning and plasmid vector construction experimentMolecular Cloning
Experimental Principles [ 123] The connection of foreign DNA and carrier molecules is DNA recombination such that recombinational DNA is called recombinant or recombinant. The recombinant DNA molecule is an exogenous DNA molecule to be linked to an exogenous DNA molecule in the connection buffer system of Mg2 + and ATP, and a carrier molecule, respectively. DNA ligase has two: T4 phage DNA ligase and E. coli DNA ligase. Both DNA ligases have functions that connect two DNA molecules with the same viscous ends, and T4 phage DNA ligase is also u0026 mdash; the characteristics of E. coli ligase can make two flat ends The double-stranded DNA molecule is connected. However, the efficiency of this connection is lower than the connection efficiency of the viscous end, generally to increase the connection efficiency of the flat end by increasing the T4 phage DNA ligase concentration or increase the DNA concentration. T4 phage DNA ligase catalytic DNA ligation reaction is divided into 3 steps: first, T4DNA ligation enzyme and adjuvant ATP formation enzyme u0026 mdash; AMP complex; then, enzyme u0026 mdash; AMP complex is incorporated into DNA having 5 u0026 RSQUO; phosphate and 3 u0026 RSQUO; hydroxyl-cutting port, DNA adenosine; then produces a new phosphate bond, seal the incision. DNA recombination method mainly has a viscous end connection method and a flat end connection method, in order to prevent the carrier itself from self-ending, can be passed (cow chineol alkaline phosphatase) CIP Treatment overcome.
The temperature of the connection reaction is conducive to the activity of the enzyme at 37 ° C. However, at this temperature, the hydrogen bond bonding in the viscous end is unstable. ThereforeA compromise temperature, namely 12-16 ° C, 12-16h (overnight), which can both exhibit the activity of the linked enzyme, and the stability of the short matching structure. 3,
Molecular Cloning u0026 Nbsp; Experimental process
The amplification and purification of DNA;
Electrophoresis detects the ligation product.
Fourth, customers provide Sample DNA, gene sequence, kit. Five, the company provides
Constructing a good carrier, electrophoretic adhesive, sequencing results.
Experimental cycle: about 1- in February.