IGEM:MIT/2007/Notebook/2007-7-24

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!

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)

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

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

Used CPX induced with AHL at concentrations: 10E (0,-5,-6,-8,-8.5)
Performed three wash steps for 15 minutes each (washed with 150 ul PBS)
Results:
- Marked difference in growth between 10E -5 CPX and negative control (DH5-alpha without transformed CPX)