The procedure below is for cloning a fragment produced by PCR into pKLAC2 . This example assumes that the PCR fragment contains a 5' XhoI site, and has a stop codon, followed by a NotI site incorporated into its 3' end.
- Prepare a PCR fragment of the gene of interest.
- Digest 0.5 μg of pKLAC2 DNA with 10 units of XhoI and 10 units of NotI in 20 μl of 1X NEBuffer 3 (supplied at 10X stock) supplemented with 100 μg/ml BSA (supplied as a 100 mg/ml stock) at 37oC for 2 hours.
- Digest 0.5 μg of the PCR fragment with 10 units of XhoI and 10 units of NotI in 20 μl of 1X NEBuffer 3 supplemented with 100 μg/ml BSA at 37oC for 2 hours.
- Add an equal volume of phenol:chloroform (1:1, v/v) to the restriction digests, mix and remove the aqueous (top) phase to a fresh tube. Repeat using only chloroform.
Alternatively, the DNA fragments can be isolated using one of the many commercially available fragmentation purification kits. If a kit is used, skip to step 8.
- Add 10 μg glycogen or tRNA as carrier to both digests, then add a 1/10th volume 3M sodium acetate, mix and add an equal volume of 100% isopropanol. Incubate at room temperature for 10 minutes.
- Microcentrifuge at 12,000 x g for 15 minutes. Pour off the supernatant and gently rinse the pellet with 70% ethanol. Allow the pellet to air-dry (~10 minutes).
- Resuspend each sample in 25 μl of 10 mM Tris-HCl, 1 mM EDTA (pH 8.0).
• 2 μl pKLAC2 digest (~40 ng)
• 10 μl PCR fragment (insert digest) (~200 ng)
• 2 μl 10X T4 DNA Ligase Buffer
• 14 μ deionized water
• 1 μl (~400 units) T4 DNA Ligase (NEB #M0202)
- Incubate at 16oC for 2 hours to overnight.
Completed ligation reactions can be stored frozen at -20oC indefinitely prior to transformation.
- Transformation of frozen competent NEB 5-aplha F' lq Competent E. coli (NEB #C2992) is recommended using 2-4 μl of the ligation reaction.
Any competent E. coli strain can be used. Blue-white screening, however, is not possible with pKLAC2.
- Prepare miniprep DNA from several transformants. Digest each vector with an appropriate restriction endonuclease to determine the presence of a cloned insert.
Expression vectors may be stored at -20oC indefinitely.