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 constructe & the 3 controls: grow them with/without arabinose to saturation in | For all 16 constructe & 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> | ||
- incubate at 37C without shaking for 30mins-1hour<br> | - incubate at 37C without shaking for 30mins-1hour<br> | ||
- check | - spin down cells for 1 min at full speed to pellet cells | ||
- check in UV box for bright white color | |||
<br><br> | <br><br> | ||
Controls:<br> | Controls:<br> | ||
Revision as of 10:42, 20 April 2009
Checks if cells do/don't bind streptavidin
For all 16 constructe & 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.
