Boston University 2006:2006-9-07

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=Thursday 7 September 2006= We grew up M30109 last night and I removed it from the inc at 11:25 am this morning. Next, we need to culture it in LB Amp+Clm broth, miniprep it, and then electroporate it into EnvZ- and WT strains of E. coli. ==Culturing== # we need to make LB stock broth (2 500 ml bottles) # we need Ampicillin stock # we need Chloramphenicol stock Then we can make 5ml of LB Amp+Clm broth in a 5ml tube to culture our E.coli containing M30109. ===protocols=== See this [http://openwetware.org/wiki/Bacterial_cell_culture OWW protocol on making cultures] for reference when making the broth. I'll post a little protocol for [[Boston_University_2006:making antibiotic stocks|making antibiotic stocks]] later. ==Glycerol Stock== It would be advantagous to preserve an amount of the M30109 culture (grown from a single colony of E. coli containing M30109 up in LB Amp+Cml broth) for posterity and as a defense against any mistakes we may make in the future. We can do this by making a glycerol stock. '''Use the Endy lab protocol''' for now because we have 40% glycerol on hand (the 100% the Quigen protocol calls for is so viscious it's hard to work with). ===protocols=== * The [[Boston_University_2006:Quigen Glycerol stock protocol|Quigen Glycerol stock protocol]] uses straight-up 100% glycerol. * There's also [http://openwetware.org/wiki/Endy:Making_a_long_term_stock_of_bacteria one] on OWW from the Endy lab. ==Miniprep== ===protocols=== [http://openwetware.org/wiki/Miniprep Miniprep protocols listed on OWW] ==Electroporation== ===protocols=== We used [http://openwetware.org/wiki/Knight:Electroporation Tom Knight's electroporation protocol] last time and it worked great.

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