Seamless L i gation Cloning Extract

SLiCE utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. This method avoids some of the pitfalls of conventional DNA cloning strategies, such as needing the appropriate restriction sites and leaving unwanted sequences at the junction sites.
Original SLiCE paper

We are using an improved and more efficient protocol developed by the Motohashi Lab, which extends SLiCE to other common lab strains and optimizes the reaction conditions.
Motohashi Lab Page
Ref [1] [2] [3]

  1. Prepare SLiCE from DH5α
    1) Pick single DH5α colony into 1mL LB and incubate on the shaker at 37℃ for 3 h
    2) Transfer culture into 50mL 2xYT and incubate on the shaker at 37℃ for 5h. Measure OD600. (If OD600 doesn’t reach 2-3, incubate overnight at 37℃. Transfer 1mL culture into another 50mL 2xYT and incubate on the shaker at 37℃ about 5h. )
    3) Harvest cells at OD600=2-3. Centrifuge at 3,000g for 15min at 4℃.
    4) Wash in 50mL ice-cold Milli-Q water. Centrifuge at 3,000g for 10min at 4℃.
    5) Resuspend 0.3-0.4 g of cells (wet weight) in 1.2mL 50mM Tris-HCl (PH=8.0) with 3% Triton X-100 (W/V) (0.2μm filtered). 6) Incubate it for 10min at room temperature. Centrifuge at 15,000g for 5 min at 4℃.
    7) Place the supernatant on ice and add 1 volume of ice-cold 80% glycerol (v/v).
    8) Aliquot 40uL of each SLiCE extract into a 0.2mL-PCR tube. Snap-freeze in a bath of liquid nitrogen.
    9) Maintain this stock solution at -80℃.

  2. 10 x SLiCE buffer (0.2μm filtered), -20℃ stock

    • 500mM Tris-HCl (pH7.5)
    • 100mM MgCl2
    • 10mM ATP
    • 10mM DTT
  3. SLiCE reaction
    vector (10-100ng) + insert DNA (insert : vector=1:1 to 3:1)
    insert DNA

    • vector DNA
    • 10xbuffer 1uL
    • SLiCE 1uL
    • add H2O         up to 10uL
    • 37℃ for 10-60 min

For help, ask Chunmei