SUSTC/22 July 2013



We had a tight schedule today for we were undertaking some key procedures in the gene engineering. One of awkward situation was that the canteen of the university had moved to the other side as well, which meant we might have to starve. However, it was apparent that we would not make hunger torment us. We had a happy lunch meeting in a small restaurant nearby.

1.Preservation of the bacteria.

Bacteria must be kept in the -80’c circumstance dissolved in the glycerol, and the content of the glycerol is roughly 20% to 30%.
Materials: 50% glycerol, bacteria in the fluid culture.
Aseptic technique:
1.absorb 600ul bacteria solution and 500ul 50% glycerol, and inject them into the 1.5ml EP tube.
2. Use the marker pen to put down the specific type and relevant resistance of the bacteria, then seal the tube and preserve in the -80’c.

2.Extract the target DNA.

Materials: bacteria(in the liquid culture), reagent case.
1. After centrifuging the bacteria, collect them in the same EP tube for many times, then leave the supernate out. 12100r/min every time, and last for 1min.
2.Inject 250ul S1 buffer into the bacteria sediment, then make them in suspension.
3.inject 250ul S2 buffer(contain RNA polymerase)(alkaline), put the EP tube upside down to split them.
4.Inject 350ul S3 buffer(acidic), put the EP tube upside down gently, then centrifuge them for 10 mins.(the time break between process 3 and 4 cannot be longer than 5 mins.)
5.Absorb the supernate and inject them into the pilar, then centrifuge for 1min and desert the waste.
6.Inject 500ul W1 buffer, then centrifuge for 1min.
7.Inject 700ul W2 buffer, then centrifuge for 1min.
8.Change the waste tube into EP tube. Then inject 30ul ddH2O into the pilar, then stew it and centrifuge for 1min.Eventually put it into the -80’c refrigerator.