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)

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2) SLiCE

The SLiCE method is a seamless cloning method and Professor Ken Honda of Kyoto Sangyo University developed more economical SLiCE method. 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 the SLiCE method, seamless cloning can be performed using extract of usual E. coli strains used in the laboratory. Because no special E. coli is used to prepare the extract, the cost per reaction is kept quite low.