Team:Georgia State/Notebook/july

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Alisha/Merhawi, Group A

Transformation using chemical competent cells
7/18/13
Page 2 Merhawi's notebook
Concentration of pGAPzaB=5ng/ul
Competent cells from "AM 3/19/13"
Protocol
1. Mix 2ul of pGAPzaB DNA with 40ul of chemically competent cells
2. Put on ice for 10 minutes
3. 42°C water bath for 30 sec
4. Put on ice for 10 minutes
5. Add 800ul SOC
6. Recover overnight in shaking incubator @ 37°C
7. Plate next day on selective media
8. After plating, let sit in gel side for 45 minutes before plating into incubator

Miniprep of pGAPzaB
7/19/13
Page 111 Alisha’s notebook
Overnight culture made by Reza 7/18/13
Axygen
Protocol
1. Collect 4 ml of overnight LB culture. Centrifuge at 12,000xg for 1 min to pellet the bacteria. Decant or pipette off as much of the supernatant as possible. (repeated step four times taking 1 (1000ul) of culture)
2. Resupend the bacterial pellet in 250ul of Buffer S1 (in fridge) by vortexing until pellet is resupended in solution.
3. Add 250ul of Buffer S2, invert tubes 6 times.
4. Add 350ul of Buffer S3 inverting 8 times. Centrifuge at 12,000xg for 10 minutes.
5. Place a Miniprep column into an uncapped 2ml Microfuge tube. Transfer the clarified supernatant from step 4 into the Miniprep column. Centrifuge at 12,000 for 1 minute.
6. Add 500ul of Buffer W1 into each Miniprep column. Centrifuge at 12,000xg for 1 minute.
7. Add 700ul of Buffer W2 into each Miniprep column. Centrifuge at 12,000xg for 1 minute.
8. Add 700ul of Buffer W2 into each Miniprep column. Centrifuge at 12,000xg for 1 minute.
9. Centrifuge at 12,000xg for 1 minute.
10. Transfer the column into a clean 1.5ml Microfuge tube. Add 80ul of Eluent to the column and let stand for 1 minute at room temp. centrifuge at 12,000xg for 1 minute.
11. Store a -20°C in Merhawi’s box.

Group C

Week 8: 7/1 – 7/5
We continued performing digestions, ligations, and transformations without being able to confirm insertion of the pGAPZαC MCS linker into pGAPZαC.

Week 9: 7/8 -7/12
We continued performing digestions, ligations, and transformations without being able to confirm insertion of the pGAPZαC MCS linker into pGAPZαC.
We also performed a midiprep on pGAPZαA + MCS linker + RFP, made and filtered sterilized 1L 10% glycerol, made LB agar for chloramphenicol plates, and prepared electrocompetent E. coli.

Week 10: 7/15 – 7/20
We had problems with arching during Electrotransporation of E. coli so we switched to transforming cells chemically.

Week 11: 7/23 – 7/27
We realized that the RFP that we had inserted in pGAPZαA + pGAPZαA MCS linker last year was not the optimal version of RFP to be inserted in yeast so we transformed E. coli with the what we thought was the more optimal RFP, but we picked the RFP from the wrong kit plate.

Week 12: 7/29 – 8/2
We transformed from Kit Plate 3 N12 RFP into E. coli and performed minipreps on an overnight culture from one of the transformed colonies.