Floating Experiments

Introduction

Using Shapiro's protocol paper (Lakshmanan et al 2017) we looked at how helpful it might be in setting up the float experiment. Pages 2060-2061 detail how to grow E. coli mega cultures, under the section C, titled 'Production and Purification of Mega GVs'. Essentially, it seems the relevant steps are (quoting directly from the text):
a) 'Resuspend an aliquot of glycerol stock in 3 ml of LB medium containing 1× ampicillin, 1× chloramphenicoland 1% (wt/vol) glucose. Grow the E. coli culture to saturation (OD600 > 4).'
b) 'Prepare 100 ml of LB medium containing 1× ampicillin, 1× chloramphenicol and 0.2% (wt/vol) glucose, and inoculate 1 ml of the saturated E. coli culture into the broth. Grow at 37 °C for ~2 h until the OD600 value reaches 0.4 to 0.6.'
c) 'Induce the culture by adding 20 μM IPTG (final concentration), and grow it at 30 °C for an additional 16–24 h.'
We followed this protocol, with a few changes that were implemented mostly by Clare Hayes, an instrumental and key part of our team. For instance, if you wanted to try different IPTG concentrations, you would want to grow up a bit of extra culture, initially. In addition, the reason the protocol says to grow the cells in chloramphenicol is because unlike us, who used the C43 strain, they transformed the plasmid into a Rosetta 2 (DE3) strain. This strain has an extra, chloramphenicol resistant plasmid, that aids in the expression of rare codons for eukaryotic gene expression. Our cells do not have this plasmid, so we don't need to use chloramphenicol - in fact, using it would kill off our cells.

Team:Warwick/Experiments

Experiments

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