©2017

DNA Assembly (Gibson Assembly)

LAB PROTOCOL

DNA assembly by the Gibson assembly method (Gibson et al., 2009) can offer several advantages in comparison with more “traditional” assembly methods (digest and ligate). These include:

  • Faster turnaround times as DNA amplification of DNA fragments is carried out in a PCR reaction.

  • Avoiding introducing of “scar sequences” in junction regions between DNA parts

  • Assembly of numerous DNA parts simultaneously (up to 5-15 parts demonstrated).

  • Single step isothermal reaction allows for more convenient/faster cloning.

  • Awesome song that can sing while carrying out the procedure steps: https://www.youtube.com/watch?v=WCWjJFU1be8

The Gibson assembly reaction mix includes an exonuclease enzyme that is able to digest one strand of duplex DNA fragments ends so that it produces single stranded overhangs. Due to the presence of overlap sequences between the DNA fragments, the resulting single stranded overhangs can base pair which allows the stitching together of parts by the ligase enzyme. At the end of the reaction, the mix should include enough circularised DNA plasmids for transformation (note from personal experience: for successful transformations use high-competency cells i.e commercially bought transformation competent cells).

Procedure steps:

  1. For each DNA fragment (and vector), design a set of oligonucleotide primers that incorporate oligo “tails” (non-binding parts) of 20-30 nucleotides overlap with adjacent part.

  2. Carry out PCR reactions to amplify DNA parts and purify the resulting DNA fragments by gel extraction.

  3. Add DNA fragments in a single tube in iso-molar concentrations so that their combined mass does not exceed 100ng (or 0.02–0.5 pmol per fragment //use online calculator http://nebiocalculator.neb.com/#!/dsdnaamt ). Note from personal experience: I find it useful to use 50ng of vector DNA and work amounts from this for the rest of the DNA fragments.

  4. Use Gibson Assembly Mastermix for the assembly reaction as specified by product manufacturer. Reactions with 2-3 DNA parts can be carried out at 50C for 30 minutes. Then place reaction mix on ice until transformation step.

  5. Transform competent cells with 5 microliters of G.A. reaction mix.

References:

Gibson, D. G., Young, L., Chuang, R. Y., Venter, J. C., Hutchison, C. A., 3rd, & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Methods, 6(5), 343-345.