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 colony (30-70 colony). 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