Most commonly used plasmids contain multiple cloning sites that can be recognized by different endonucleases. Because of the large number of cloning sites available (e.g., Invitrogen's PSE280 plasmid has 46 cloning sites, and it is possible to design polycondenser junctions with more cloning sites (Brosius 1992)), it is generally always possible to find a plasmid vector with an enzyme cleavage site that matches the end of a particular exogenous DNA fragment. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Huang Peitang et al.
Operation method
Targeted cloning experiments in plasmid vectors
Principle
Most commonly used plasmids contain multiple cloning sites that can be recognized by different endonucleases. Because of the large number of cloning sites available (e.g., Invitrogen's PSE280 plasmid has 46 cloning sites, and it is possible to design polycondensers with even more cloning sites (Brosius 1992)), it is generally always possible to find a plasmid vector with an enzyme cleavage site that matches the end of a particular exogenous DNA fragment.
Materials and Instruments
T4 Phage DNA ligase Restriction endonuclease Move I. Materials For more product details, please visit Aladdin Scientific website.
ATP Ethanol Phenol Chloroform Sodium acetate TE
Agarose gel Polyacrylamide gel Rotary column chromatography device
1. Buffers and solutions
ATP (10 mmol/L), ethanol, phenol:chloroform (1:1, V/V), sodium acetate (3 mol/L, pH 5.2), TE (pH 8.0).
2. Enzymes and buffers
T4 phage DNA ligase, restriction endonuclease
3. Gel
Agarose gel, polyacrylamide gel.
4. Nucleic acids and mononucleotides
Carriers DNA (plasmid), exogenous or target DNA fragments.
5. Specialized equipment
Rotary column chromatography device, water bath with adjustable temperature of 16 ℃.
Methods
1. Digest the vector (10 ug) and exogenous DNA fragments with two appropriate restriction endonucleases.
For direct cloning, the plasmid vector was digested in a closed loop with two restriction endonucleases so that different sequences could be recognized as well as different ends produced. Regardless of the sequence, we should avoid, if possible, using sites within 12 bases of each other at the multiple cloning site; if one site is cleaved, the second site will be very close to the end of the linear DNA molecule and the efficiency of the second restriction enzyme will be compromised. The New England Biolaba catalog lists the cleavage efficiencies of various restriction enzymes at the terminal sites of linear DNA molecules.
Read the manufacturer's instructions to determine if both restriction enzymes work in the same buffer. If they do, you can use both restriction enzymes to digest the plasmid DNA at the same time. If the two restriction enzymes are in different buffers, it is best to keep the digestion reactions separate. In this case. The enzyme suitable for low salt concentrations should be used first. After the reaction with the first enzyme is completed, take a small amount of the digested product and electrophoresis it to determine if all the plasmid DNA has changed from cyclic to linear molecules, then adjust the salt concentration of the buffer appropriately before adding the second enzyme.
2. Phenol: Chloroform pumping and ethanol precipitation methods are used for purification of the digested exogenous DNA fragments.
Depending on the experiment, we can refer to agarose gel or polyacrylamide gel electrophoresis to isolate the target fragments from the exogenous DNA digestion products. This purification is generally required when the exogenous DNA fragment preparation contains multiple restriction enzyme fragments that can be ligated instead. Many researchers often use agarose gel electrophoresis to improve the purity of the exogenous target DNA sequence prior to the ligation reaction, rather than mass screening of the transformants for the desired clone.
3. Purification of vector DNA by centrifugal column chromatography plus conventional ethanol precipitation
This purification step removes from the plasmid preparation those small fragments produced by restriction enzyme digestion of two of the close restriction enzyme sites in the polyclonal locus. DNA.
4. Re-dissolve the two purified DNA precipitates in TE (pH 8.0) to a final concentration of approximately 100 ng/ml. Assuming that 1 bp is equivalent to 660 Da, calculate the concentration of DNA (pmol/ml).
The approximate concentration of the two DNA samples is checked by agarose gel electrophoresis.
5. Transfer the appropriate amount of DNA into 0.5 ml sterile microcentrifuge tubes according to the following table: 
(1) Tubes A, B, and C are filled with: 1.0 ul of 10x Ligation Buffer, 0.1 unit of T4 Ligase, 1.0 ul of 10 mmol/L ATP, and 10 ul of H2O.
(2) Tubes D and E are filled with: 10x Ligation Buffer, 0.1 unit of T4 Ligase, and 1.0 ul of 10 mmol/L ATP. Tubes D and E were filled with: 10X Ligation Buffer 1.0 ul, 10 mmol/L ATP 1.0 ul, H2O to 10 ul, no DNA ligase.
During preparation of the DNA fragments, the DNA fragments can be added to the tubes together with water and incubated at 45°C for 5 min to eliminate end cross-polymerization due to re-duplication. The DNA solution can be pre-cooled at 0°C before the ligation reagents are added. For maximum efficiency of ligation, the smaller the reaction system the better, typically 5 to 10 ul. In the reaction mixture, ATP, if used as a component of the 10X ligation buffer, provides more room for the volume of vector and exogenous DNA insert fragments. Some manufacturers include ATP in the ligation buffer so that it is not necessary to add additional ATP in the application.
6. Place the ligation mixture at 16°C overnight or 20°C for 4 h.
7. Transform the susceptible E. coli with the diluted ligation product. A known amount of superhelical plasmid DNA prepared by the standard method should be included as a control to check the transformation efficiency. 