IGEM:MIT/2007/Notebook/2007-7-24
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AGENDA
- Plate the Mer/I13500 3A transformation after overnight ligation (and grow)
- Check if sequence results are available from Monday (length was correct)
- Second polystyrene assay (JH and TTP?)
- CPX, PBS with different concentrations and controls
- Suggested: one person per plate, pipet to the edge, more washes, harder aspirations
- Sequence the FhuA (pHIE) with CORRECT primers (which already exist?)
- Prepped plasmid exists (AL, 7/23)
- EC_20 requests
- Harass the people AK e-mailed OR design and order?
- If T7 antibody comes in...test!
Transformation of Mer/I13500 after overnight ligation (in TK's DH5alpha)
- Added 5µL of ligation reaction to 100µL of DH5a
- Incubation in 37 degree room for one hour and forty-five minutes (Amp and Tet resistant)
Sequencing of FhuA (picss8)
- Used FhuA, 10 µl to make primers (top and bottom)
- 1 µl of FhuA, 10 µl + 2.12 µl water = 3.2 primer
- 1 µl of primer + 2 µl DNA + 9 µl water = 12 µl total sequencing mixture
- DNA = "picss8" with nanodrop of 99.6
- Strip of 8 PCR tubes labeled "Liying Huang, 8201" sent in for sequencing at 3 PM
- 1 = top primer
- 2 = bottom primer
Transformation of CPX (A+,Cm+), FhuA (K+) and pUC18 (A+) into Barry's BL21 cells (more protein!)
- Obtained pUC18 and BL21 courtesy of Barry
- BL21 Cell volume: 50 µl
- DNA mass to add: 50 to 60 ng
- For spreading on plate: 300 µl and let dry
- NOTE: to help plates absorb liquid bacteria, leave plates out overnight after making them before putting them in fridge (dries them out)
- "BL21 trans. w/ pUC18 (A+)": pulled out 55 µl of pUC (at 1 ng/µl of stock) and added to cells
- "BL21 trans. w/ FhuA (K+)": diluted 1 µl of stock into 2 µl total; pulled out 1.5 µl and added to Bl21
- "BL21 trans. w CPX (Cm+ A+)": diluted 1 µl of stock into 4 µl total; pulled out 1.5 µl and added to BL21