Affinity Protocol
1) Grow minicolonies in microtiter wells as describe in the basic protocol for growing minicolonies.
2) Slowly remove media from well and place in 1.5 mL microcentrifuge tube, carefully placing pipette tip on the left side of each well. With all subsequent washes, continue to place pipette tip in same spot.
3) Media + Gentle Wash culture: Add 250 uL ddH2O to the well. Remove and combine with media in 1.5 mL microcentrifuge tube. Do this wash 4x.
4) Vigorous Wash Culture: Add 250 uL ddH2O to the well after the final gentle wash. Mix cells remaining in well by vigorously pipetting up and down 10x. Remove liquid and place in a separate 1.5 mL microcentrifuge tube.
5) Spin down the culture containing Media + Gentle wash. Remove 1 mL of supernatant and resuspend cells in the remaining 250 u.
6) Sonicate both cultures 3x, 6 seconds each time.
7) Count cells with a hemacytometer and determine the percentage of total cells in the Media + Gentle Wash culture vs. the percentage in the Vigorous Wash culture.
