Growing Bacterial Cultures for DNA Plasmid Isolation

Biochemistry, Molecular Biology, and Cell Biology Protocols  >> Growing Bacterial Cultures

Growing Bacterial Cells

LB Broth
water 950mL
bactotryptone 10g
yeast 5g
NaCl 10g

alternatively, for LB broth, add 25g/L of LB powder

when using LB broth as a medium to shake in a flask, 
for 2L flasks, the maximum amount of media to use is ~500mL, or of the volume of the flask to allow for sufficient oxygen supply for the bacteria

Making up a permanent stock  of bacterial cells
830uL of freshly-grown bacterial culture
170uL of 85% glycerol
freeze in the 80-degree freezer

Growing cells from a permanent stock for maxi-plasmid preparations
1. keep the cells on a freezer block when taking them out
2. flame tweezers with bunsen burner
3. pick up sterile pipet tips with tweezers to scoop up cells
4. place in ~5mL of broth containing the desired antibiotic
5. shake overnight
6. take 500uL of the culture and place into 250mL of LB broth (in a 2L flask) to shake overnight
7. ready for DNA plasmid isolation the next day

Growing cells from a transformed plate
1. flame tweezers with bunsen burner
2. use tweezers to pick up sterile pipet tips
3. pick one colony
4. dump the pipet tip into a culture tube with 2mL LB and shake overnight at 37 degrees C

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