IGEM:University of Chicago/2009/Notebook/Paraoxon Biosensor/2009/07/06

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July 6th, 2009

Goal: Conducted transofrmation w/ new PCR product. Also ran Damon's surface display system start-up culture.

Pour Kan: YPD plates Recipe: YEP (2x) 300 ml Agar (4%) 800 ml Glucose (40%) 30 ml G418 (kan) 2.4 ml -50 mg/ml stock

1. Melt agar (microwave 4 min), tighten cap

2. Cool 10-15 min

3. Add glucose and G418 t o 2x YEP bottle, mix

4. Add 3) to cooled agar mix

5. spray 95% EtOH to surface to get rid of bubbles

6. pour immediately onto plates, let cool

7. When solidified, mark plates (3 blue line), put into bag, store at 4 degrees C

Transformation (20 ml total ~ 8 transformations) 1. 10+10 ml culture incoulated w/ Robs O/N from 6/30/09...began @ 11 a.m.

2. reached OD of 0.255 (660 nm) at 4:30 p.m....~ 5 hours...longer growth due to old culture

3. Spin down

4. Add 4ml sterile TF (?) Spin, decant

5. Add 4 ml LiAC mix. Spin, decant

6. Resuspend in 200 ul LiAc

7. 7 tubes: 2ml miniprep DNA in respective eppendaef tube. +25 ul suspension mix in each. + 200 ul PEG mix to each tube and vortex

8. incubated 30 degrees C (30 min)

9. heatshocked in 42 degrees C water Bath (20 min)

10. pelleted yeast

11. Re-suspend in 200 ul SoS

12. Plated 200 ul of solution to these respective labeled Kan Plates

13. placed in incubates (30-31 degrees C)

End of the day