Template:SBB-Protocols Assay2: Difference between revisions
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Checks if cells do/don't bind streptavidin<br> | Checks if cells do/don't bind streptavidin<br> | ||
For all 16 | For all 16 constructs & the 3 controls: grow them with/without arabinose to saturation in 1-3uL culture (grown in 24 well blocks)<br> | ||
96 well plate: <br> | 96 well plate: <br> | ||
- each well add 300ul PBS, 15ul Streptavidin, 25ul of cell culture (the first time we do this, it will be saturated culture) <br> | - each well add 300ul PBS, 15ul Streptavidin, 25ul of cell culture (the first time we do this, it will be saturated culture) <br> | ||
| Line 20: | Line 20: | ||
<font color="darkturquoise">JCA: This needs much more detail. In particular, write up the details about the volumes of materials, composition of the assays, times and temperatures at each step, etc.</font> | <font color="darkturquoise">JCA: This needs much more detail. In particular, write up the details about the volumes of materials, composition of the assays, times and temperatures at each step, etc.</font> | ||
''April 22'' | =='''April 22'''== | ||
- today we tried to do the assay with 25ul cells in 300ul PBS with 15ul Streptavidin<br> | |||
- we could actually visually see binding for 4 constructs (one control?)<br> | |||
- we tried to quantify it with the Tecan but we just got background<br> | |||
'''for Monday'''<br> | |||
- try 200ul cells<br> | |||
- spin down<br> | |||
- discard the media<br> | |||
- resuspend in 200ul PBS<br> | |||
- use 5ul Streptavidin<br> | |||
- incubate at 37 for 30 mins<br> | |||
- spin down, remove supernatant<br> | |||
- check visually<br> | |||
- resuspend in 50 ul of PBS and load on plate for the Tecan<br> | |||
- analyze measurements <br> | |||
Revision as of 13:06, 22 April 2009
Checks if cells do/don't bind streptavidin
For all 16 constructs & the 3 controls: grow them with/without arabinose to saturation in 1-3uL culture (grown in 24 well blocks)
96 well plate:
- each well add 300ul PBS, 15ul Streptavidin, 25ul of cell culture (the first time we do this, it will be saturated culture)
- incubate at 37C without shaking for 30mins-1hour
- spin down cells for 1 min at full speed to pellet cells
- check in UV box for bright white color
Controls:
pBca9145-Bca 9494 (positive control that displays cpx, a streptavidin binding peptide under Pbad)
DH10B (no plasmid, negative control)
pBca9495CA-Bca1144 (negative control: backbone)
Can play with:
- streptavidin concentrations
- mid-log vs. saturation
- quantitative assays (number of washes, volume...)
JCA: This needs much more detail. In particular, write up the details about the volumes of materials, composition of the assays, times and temperatures at each step, etc.
April 22
- today we tried to do the assay with 25ul cells in 300ul PBS with 15ul Streptavidin
- we could actually visually see binding for 4 constructs (one control?)
- we tried to quantify it with the Tecan but we just got background
for Monday
- try 200ul cells
- spin down
- discard the media
- resuspend in 200ul PBS
- use 5ul Streptavidin
- incubate at 37 for 30 mins
- spin down, remove supernatant
- check visually
- resuspend in 50 ul of PBS and load on plate for the Tecan
- analyze measurements
