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1)Boil Method

1.Monitor OD of yeast and incubate until it becomes OD≒1.
2.Centrifuge yeast at 3500 rpm for 5 min
3.Discard the supernatant
4.Pipette the culture residual substance and transfer 1 ml of it to an Eppendorf tube
5.Centrifuge on FLASH and then decantate it
6.Add 1 ml of distilled water
7.Vortex
8.Centrifuge yeast at FLASH
9.Discard the supernatant (wash 1st time)
10.Repeat steps 6-9 (wash 2nd)
11.Repeat steps 6-9 (wash 3rd)
12.Cryopreservation
13.Add 1 ml of distilled water and vortex
14.Boil it with hot water for 10 min
· Fit the tube in the sponge and put it in boiling water
(· Keep the fire between low to medium heat)
15.Centrifuge at 10000 rpm for 5 min, and remove 900μl of the supernatant
16.Measure Na+ concentration(Atomic Absorption Spectrometry)

2) SLiCE(Seamless Ligation Cloning Extract)

The SLiCE method is a seamless cloning method. The original one is requied special E. coli with high endogenous in vitro homologous recombination activity. Professor Ken Honda of Kyoto Sangyo University optimized the reaction condition and developed more economical SLiCE method in which special E. coli is not nessesary. Cloning kits that use the seamless cloning method, such as In-Fusion and Gibson Assembly, have been commercialized variously, but many of the kits are expensive, especially for student organizations like us. In this SLiCE method, seamless cloning can be performed using extract of usual E. coli strains used in the laboratory. Thanks to this inexpensive seamless cloning method, we succeed in cloning of our important parts: SseNHX1 and ZrGPD1. In this case, overlaps between inserts and vectors were 30bp, though we could get enough colonies (30-70 colonies). If you follow the protocol below, you can probably conduct more efficient cloning. We hope this convenient cloning method is propageted all over the iGEM!

Figure1. SLiCE method

1.Preparation of SLiCE(from JM109)
JM109 stored at -80℃
　　　　※DH5a, XL10-Gold, Mach1-T1R can be used
　　| 1mL LB is maintained at 37℃, usually for 3 h.
　　| 50mL 2xYT is maintained at 37℃, usually for 4-5 h.
　　Harvest the cells at OD = 2 to 3.
　　　※If OD does not reach to 2-3, incubate overnight at 37℃.
　　　　　Next day, transfer 1mL overnight culture into another 50mL 2xYT and incubate at 37℃ for several hours.
　　Centrifuge at 5,000xg for 10min at 4℃.
　　Wash in 50mL ice-cold Milli-Q water.
　　Centrifuge at 5,000xg for 5min at 4℃.
　　0.3-0.4 g of cells (wet weight)
　　Resuspend gently in 1.2mL CelLytic B Cell Lysis Reagent (Sigma, B7435) .
　　　　※Alternatively, buffers containing Triton X-100 can also be used.
　　Incubate it for 10min at room temperature.
　　　　※Leave to stand, not rorate.
　　Centrifuge at 20,000xg for 2min at 4℃.
　　Place the supernatant on ice and add 1 volume of ice-cold 80% glycerol (v/v).
　　Aliquot 40uL of each SLiCE extract into a 0.2mL-PCR tube.
　　Snap-freeze in a bath of liquid nitrogen.
　　Maintain this stock solution at -80℃ (for use, 40uL each extract in 0.2mL 8-strip PCR tube).
　　　　※SLiCEs can be stored at -80℃ for at least three years without significant loss of activity.
　　　　※For short-term storage, SLiCEs are stored at -20℃.
　　　　※SLiCEs can be stored at -20℃ for 2-3 months without significant loss of activity.
2. 10x SLiCE buffer(0.2µm filtered), -20℃ stock
500mM Tris-HCl(pH7.5)
100mM MgCl2
10mM ATP
10mM DTT
3.Vector
PCR amplified vector or restriction enzyme-digested vector
4.Insert DNA
Design primer for amplification of insert. Make sure overlap regions be 19bp.
Use high fidelity polymerase when you amplify insert.
5.Protocol
･PCR (19bp overlap sequence)
･Purification of PCR fragments (by silica column)
　　　　　If multiple bands were amplified by PCR, the PCR fragments should be purified by agarose gel electrophoresis.
･vector(10-100ng) + insertDNA (insert:vector=1:1 to 3:1)
　　　　　　※Excess insert DNA inhibits the SLiCE reaction.
･SLiCE reaction
　　　　　InsertDNA
　　　　　VectorDNA
　　　　　10xB 1uL
　　　　　SLiCE 1uL<>br 　　　　　DDW 　　　　　　　　　　　　　　　up to 10uL
　　　　　37℃, 15 min (Longtime incubation reduce colony formation rate.)