BioBrick to cell culture
This protocol specifies how to create a cell culture from a BioBrick order. The culture can then be miniprep-ed and/or made into freezer stock.
- Starting with BioBrick order, streak (using toothpick or pipet tip) onto an amp (or other antibiotic, as appropriate) LB selective plate. Grow overnight at 37°C.
- Pick a single colony with sterile toothpick and transfer the colony to a culture tube that has 5 mL of LB broth with antibiotic (1:1000 for amp, or 5 µL). If you are making multiple stocks, it is helpful to make a broth/antibiotic master mix.
- You could take a fresh bottle of LB, add the correct amount of antibiotics, and store the stock at -20°C. That way, you don't have to thaw the antibiotics each time.
- Put in 37°C shaker (at approximate speed 180) and let grow overnight.
- For BioBricks in pSB2K3 plasmid, the cells need to grow in 5 mM IPTG solution for 3 h. The 500 mM IPTG stock can be found in the fridge in the box with Amp stock.
- Another strategy to increase DNA yield is to back-dilute to one-tenth before adding IPTG.
- Another protocol for pSB2K3, found here says: "When using pSB2k3, you must add IPTG to induce the high copy origin of replication. Once culture has grown up, add 10 μl of 0.8M IPTG for each 5ml of culture. Incubate for an additional 4-5 hours. Make glycerols before the addition of IPTG."
- From the parts MIT site, "Induction to high copy can be carried out by addition of IPTG (1mM final concentration). IPTG can be added at different points during growth (to suit your application). For instance, to prepare both cells (e.g., for glycerol stocks) and DNA (e.g., for further cloning) grow the cells to mid log, pull a sample for the glycerol stock, add IPTG and continue growth, prepare DNA from a saturated culture."