By Rifei Chen

For the linear backbone were so few and the backbone of BBa_J04450 was pSB1C3, we decide to cut it by XbaI and PstI restriction enzyme and recycle the about 2000bp vector as the backbone for biobricks. Of course, one advantage to make it as backbone is that we can recognize whether the gene we designed have inserted. Because if not, the bacterium will express red chromoprotein which one we unnecessary to pick up.

week 4, Aug

2014/08/22

Transfect the backbone of biobricks

1. Add 10μl ddwater to the plate, wait 10min, and absorb all the liquid into a PCR tube.
2. Took a tube of supercompetent cells from -80℃ fridge, Immediately place the tube on ice, allow cell to thaw on ice, 10 min.
3. Check the cell to see if they have thawed, gently flick the cells 1-2 times to evenly resuspend the cells.
4. Mark the label with BBa_J04450.
5. Add 2μl plasmid to the tube, shaking slightly.
6. Incubate on ice for 30min.
7. Heat shock, 42℃, 90s.
8. Keep on ice immediately, 2 min.
9. Add 200μl LB medium to each tube, shake with 37°C, 200rpm, 45min.
10. Centrifuge, 4000rmp, 3min at room template.
11. Discard most of the medium and reserve 50μl, resuspend by tips.
12. Flam the SS-Spreader; put aside to cool; coated plates.
13. Incubate the plates at 37°C, 16 hours.

2014/08/23

Shake the bacterium in tubes

1. Add 3ml LB broth with ampicillin antibiotics into a tube.
2. Flam the tweezers to red; put aside to cool.
3. Pick up a tip which was sterilized; pick monoclone and throw the tip with monoclone into the tube directly.
4. Shake with 37°C, 200rpm, and overnight.