From 2006.igem.org

Contents 1 Thursday 7 September 2006 1.1 Culturing 1.1.1 protocols 1.2 Glycerol Stock 1.2.1 protocols 1.3 Miniprep 1.3.1 protocols 1.4 Electroporation 1.4.1 protocols

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

1. we need to make LB stock broth (2 500 ml bottles)
2. we need Ampicillin stock
3. 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 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 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.