Structural Biochemistry/Cloning Tips
Some general considerations in the process of cloning can enhance the efficiency of cloning. The following are some tips:
1. The DNA insert/PCR product and vector digested by appropriate restriction enzymes should be purified directly, or from an agarose gel. This gets rid of the primers or the restriction fragments that are not of interest.
2. When performing ligation, the insert:vector ratio should be tested for the optimal conditions. Generally, a 1:1 or 3:1 ratio works well.
3. Screening for correct clones containing the vector with the DNA of interest inserted is very important. Common methods such as blue/white screening for B-Galactosidase activity and antibiotic screening are applied.
4. Calf Intestinal Alkaline Phosphatase is a hydrolase enzyme that is responsible for removing phosphate groups from nucleotides, proteins, etc. This enzyme can be used after digestion with restriction enzymes.
5.ATP is required for ligation.
6. Enzymes are used to synthesize, degrade, join or remove portions of nucleic acids in a controlled and generally defined manner.
7. Cloning Enzymes, are those important in nucleic acid cloning procedures. Some cloning enzymes are used as ligases, kinases and phosphatases, and RecA proteins.
8. When using vectors with antibiotic resistance characteristics for selection, after transforming the cells allowing the cells to grow in appropriate growth medium and growth temperatures for several hours increases growth efficiency as well as antibiotic-resistance. This will allow for cells that successfully took up the cloned vectors to be better collected once plated onto antibiotic treated growth plates. The preliminary growth period in solution will stimulate cell activity in producing the antibiotic resistance characteristics.
9. Cross contamination when working with multiple cloned DNA vectors with similar antibiotic selections is a concerning issue. Washing, ethanol-treating, and flaming instruments is an important anti-cross-contamination measure to be taken seriously.
10. Sterile technique.