Morning
Performed Calibration 1, 2, and 3 according to protocol. All measurements were performed using the plate reader of Seino.
Table 2: Receptor EC50 values
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
|---|
| A | L | dd |
| B | L | dd |
| C | L | dd |
| D | L | dd |
| E | MS | dd | dd | dd | dd | dd | dd | dd | dd | dd | dd | dd |
| F | MS | dd | dd | dd | dd | dd | dd | dd | dd | dd | dd | dd |
| G | MS | dd | dd | dd | dd | dd | dd | dd | dd | dd | dd | dd |
| H | MS | dd | dd | dd | dd | dd | dd | dd | dd | dd | dd< | dd |
Calibration 1
L= 100 uL Ludox CL-X (stored at 4C)
dd= 100 uL ddH20
Measurement: Abs600, turn off pathlength correction
Calibration 2
MS= 200 ul Microsphere Stock Solution
dd= 100 uL ddH20
green= serial dilution was performed with a micropipet from E1,F1,G1,H1 - E11,F11,G11,H11 by a volume of 100uL. Before every transfer solution was pipetted up and down 3x, after every transfer tips were discharged.
Measurement: Abs600, re-mix befor putting in plate reader and prevent bubbles, path length correction off
Calibration 3
1xFC= 200 mL 1xFC (100uL 10x fluorescein + 900ul 1x PBS pH 7.4, tube was covered with foil
P= 100 uL 1x PBS pH 7.4
green= serial dilution was performed with a micropipet from A1,B1,C1,D1 - A11,B11,C11,D11 by a volume of 100uL. Before every transfer solution was pipetted up and down 3x, after every transfer tips were discharged.
Measurement: FL, 530nm/30nm bandpass, 25-30nm with recommened excitation of 485nm, emission 520-530nm of the filter. Path length correction was turned off
07/08/18 (afternoon)
LBC plates were made according to the protocol used on the wall
250ml LB 2x added to melted 250 ml WA 2x using a microwave
0.5ml was added to final solution
plates were dried in 37C incubator
Transformation device 3 + negative control interlab study
Device 3 (number 5) showed a low GFP expression, so it was tried to re-preform the tranformation. Negative control of the interlab (number 1) was not performed last time due to lack of LBC plates so was also performed.
protocol used:
competent cells (DH5-a) was put on ice for 20 min
50ul of competent cells were transferred to inoculation tube, one for each separate DNA sample
2ul DNA (number 5 or 1) was added
30 min on ice
42 C heatshock for 45 sec
coldshock on ice 5 min
500uL SOB was added to each of the tubes
1h incubated in 37C shaker
plated on LBC plates and incubated overnight at 37 C
100ul directly (with resuspending)
centrifugation at full speed for 30 sec
300ul was removed
pellet was resuspended in remaining 100ul
100ul was plated onto LBC plate
