Triplicate extracts same culture
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Background: Different batches of extract show wide variability. It is well documented that the harvest OD, lysis method, run-off time, and dialysis time play a crucial role in extract variability. But are these all the major sources of variability?
Experiment: 3 technical replicates of extract were made in parallel (by 3 people working at once) from 6L of E. coli culture which was remixed together after being removed from the shaker but before centrifugation. The Jove protocol (link on main page) was used in this experiment. Each batch resulted in similar amounts of final extract. Then, the maximal fluorescence output during salt calibration (Mg-Glutamate x K-Glutamate panel) of the three extracts (performed with a liquid handling robot) and measured in a Biotek plate reader.
Results: The maximal fluorescent output of each replicate varied widely. The optimal salt concentration varied less widely. Sources of variability are unknown but presumably must involve: centrifugation, decantation, or lysis (with a french press).
