Enzyme Free Cloning

Protocol

- order primers for each gene of interest (...) with the following termini:

short forward 5’ TG...

LIC forward 5’ GCCGCGCGGCAGCCTG...

short reverse 5’ TCA...

LIC reverse 5’ CAAGAAGAACCCCTCA...

- Perform PCR A with LIC forward and short reverse primer

- Perform PCR B with LIC reverse and short forward primer

- If you use plasmid DNA as template for the PCR, remove the input plasmid DNA by treatment with the methylation specific restriction enzyme DpnI (37 �C, 30 min) or use a maximum of a nanogram of plasmid DNA template. This step is not required when amplifying cDNA.

- After the PCR, purify the PCR products according to the guidelines by the manufacturer of your PCR cleanup kit/beads. In our hands, Agencourt Ampure magnetic beads work fine.

- After the purification, mix 5 ul of PCR A and 5 ul of PCR B in a PCR plate/tube, incubate for 5’ at 95C in the PCR machine and let the products cool down to room temperature in the machine without a fast cooling gradient (takes appr. 15 minutes).

- Add 1.0 ul of the vector stock to appr. 2ul of to the mix of PCR(A+B) and incubate for 5 minutes at room temperature. If your PCR is hardly visible on gel, add 5 times more PCR product. 1.0 ul vector should contain approximately 1-3 ng of T4 treated vector when using chemically competent E. coli cells with a competence of >5E+05 col/ug.

- Add 20-25 ul of competent E. coli (more cells if >5ul hybridization mix is used) and perform standard transformation.

Notes: ALWAYS take along a negative control of vector without adding any insert! Preferably, take along as a positive control a PCR product that worked previously. Typically, 1-2 ng of vector gives approximately 100 colonies using E. coli with a competence of 1E+06 col/ug.

Modifications: you can eliminate the need for two primer sets for each gene by using LIC adaptor primers that recognize the termini of all your PCRs. They can, however, generate primer-dimer artifacts, so the above method seems superior. Rob cools down to room temperature in 5 minutes with comparable results and uses 15 ul of cells with 3 ul of hybridization mix. It is advisory to keep the volume of hybridization mix < 25% of the competent cell volume to prevent comprimizing the chemical competence. Alternatively, you could add CaCl2 to compensate for your extra volume.

References:

Liu, Z. (1996) Hetero-stagger cloning: efficient and rapid cloning of PCR products. Nucleic Acids Res, 24, 2458-2459.

Shih, Y.P., Kung, W.M., Chen, J.C., Yeh, C.H., Wang, A.H. and Wang, T.F. (2002) High-throughput screening of soluble recombinant proteins. Protein Sci, 11, 1714-1719.

Tillett, D. and Neilan, B.A. (1999) Enzyme-free cloning: a rapid method to clone PCR products independent of vector restriction enzyme sites. Nucleic Acids Res, 27, e26.

Zeng, G. (1998) Sticky-end PCR: new method for subcloning. Biotechniques, 25, 206-208.

R.N. de Jong, M.A. Dani�ls, R. Kaptein and G.E. Folkers (2007) Enzyme Free Cloning for high throughput gene cloning and expression. J. Struct. Funct. Genomics, in press.