In order to construct new DNA molecules, the starting DNAs are treated with appropriate restriction endonucleases and other enzymes if necessary. The individual components of the desired DNA molecule are purified by agarose or polyacrylamide gel electrophoresis,combined, and treated with DNA ligase.

The products of the ligation mixture (along with control mixtures) are introduced into competent E. coli cells, and transformants are identified by an appropriate genetic selection. DNA is prepared from the colonies or plaques and subjected to restriction endonuclease mapping in order to determine if the desired DNA molecule was created. All cloning experiments follow the steps outlined below.

Materials

Calf intestine phosphatase (CIP) and buffer	dNTP mix (0.5 mM each; )
Klenow fragment of E. coli DNA polymerase I or T4 DNA polymerase	Oligonucleotide linkers (optional)
10 mM ATP	0.2 mM dithiothreitol (DTT)
T4 DNA ligase	2×T4 DNA ligase buffer

Protocol

1. In a 20-μl reaction mixture, cleave the individual DNA components with appropriate restriction endonuclease. After the reaction is complete, inactivate the enzymes by heating 15 min to 75°C. If no further enzymatic treatments are necessary, proceed to step 6.

2. If the 5′ phosphates of one of the DNAs are to be removed, add 2 μl of 10× CIP buffer and 1 U CIP; incubate 30 to 60 min at 37°C. After the reaction is complete, inactivate CIP by heating 15 min to 75°C. If no further enzymatic treatments are necessary, proceed to step 6.

3. If one or both ends generated by a restriction endonuclease must be converted to blunt ends, add 1 μl of a solution containing all 4 dNTPs (0. 5 mM each) and an appropriate amount of the Klenow fragment of E. coli DNA polymerase I or T4 DNA polymerase; carry out the filling-in or trimming reaction.After the reaction is complete, inactivate the enzymes by heating 15 min to 75°C. If oligonucleotide linkers are to be added, proceed to step 4. If a DNA fragment containing only one blunt end is desired, cleave the reaction products with an appropriate restriction endonuclease. If no further enzymatic treatments are necessary, proceed to step 6.

4. Add 0. 1 to 1. 0 μg of an appropriate oligonucleotide linker, 1 μl of 10 mM ATP, 1 μl of 0. 2 M DTT, and 20 to 100 cohesive-end units of T4 DNA ligase; incubate overnight at 15°C. Inactivate the ligase by heating 15 min to 75°C.

5. Cleave the products from step 4 with the restriction endonuclease recognizing the oligonucleotide linker, adjusting the buffer conditions if necessary. If only one of the two ends is to contain a linker, cleave the products with an additional restriction enzyme.

6. Isolate the desired DNA segments by gel electrophoresis, or by other methods if appropriate.

7. Using longwave UV light for visualization of the DNA, cut out the desired band(s) and purify the DNA away from the gel material using the procedures described.

8. Set up the following ligation reaction:9 μl component DNAs (0. 1 to 5 μg)    10 μl 2×ligase buffer    1 μl 10 mM ATP    20 to 500 U (cohesive end) T4 DNA ligase     Incubate 1 to 24 hr at 15°C.

9. Introduce 1 to 10 μl of the ligated products into competent E. coli cells and select for transformants by virtue of the genetic marker present on the vector.

10. From individual E. coli transformants, purify plasmid or phage DNAs by miniprep procedures and determine their structures by restriction mapping.