Transformation of plasmid DNA into competent cells
Before beginning, remove LB agar plates with appropriate antibiotic and place in 37°C incubator to warm up.
Pre-chill sterile 2ml microcentrifuge tubes on ice (one for each plasmid)
Thaw one tube of XL-10 Gold Ultracometent cells on ice (~15-20 minutes)
When thawed, gently mix (for this volume, you can gently flick) and aliquot 25ul (Lars used 10 ul, it was enough and worked well) of cells into each tube. Remaining cells can be returned to -80°C. The cells should always be on ice if not in the freezer!
Add 1ul of β-ME to each aliquot of cells. (the ratio is 1:25, so 4ul for 100ul of cells).
Mix the tubes by gently flicking. Incubate cells on ice for 10 minutes, flicking tubes gently every 2 minutes.
Add 1ul of plasmid DNA to each aliquot of cells. (If you are drawing from a tube with only a few microliters inside, spin the tubes down first in a minifuge). Make sure you label your tubes if you are transforming multiple plasmids! Mix (or flick) gently then incubate on ice for 30 minutes.
Heat-pulse the tubes for 30 seconds in 42°C water bath or heat block filled with water. If using heat block, add the water at least 5 minutes prior so it will warm to 42°. The duration of the heat pulse is critical, time it exactly.
Immediately move cells to ice for 2-minute recovery incubation.
**All operations involving LB must be performed with an open flame. For maximum safety remove gloves before lighting and rub hands with 70% EtOH
Add ~1ml of LB (RT or pre-heated, but not cold) to each tube. Incubate for 1 hour at 37°C with shaking (200-250 rpm). This step is not critical, some people skip it. But if you skip it your cells will grow more slowly. If you can do an incubation for at least 15 minutes it will speed up cell growth.
Plate transformation mixture on pre-warmed LB agar plates containing appropriate antibiotic (usually Ampicillin/Carbenicillin). For Line 1 plasmids in XL-10 gold cells, 10-50ul of transformation mixture is more than enough. Save remaining cell mixture at 4°C and re-plate the next day if necessary. You can use glass plating beads or an inoculation loop for plating. Plate "blank" water control to detect contamination.
Incubate plates at 37°C overnight. Remove plates the next day and seal with parafilm. They can be stored for a day at RT on your bench or at 4° for long periods of time.
Inoculate Bacterial Culture
With an open flame, prepare an Erlenmeyer flask for each plasmid. Choose flasks that are double the volume of your culture, to allow for proper aeration while shaking. Use 1000ml flasks for 250ml culture volume. Label the flasks with appropriate plasmid name before beginning.
Add 250ml LB to each flask, then add 100ug/ml of Ampicillin. Ampicillin stocks are stored at -20C and are usually 100mg/ml. So you should add 250ul of the stock to each flask.
Pick a single colony from the plate with a pipette tip and drop it in the flask. Make sure you are matching the plasmid #s between plate and flask.
Cover each flask with foil (flame the foil first) then place in 37°C shaking incubator (200-250 rpm) overnight.
Midi Prep
After 18-24 hours, remove the flasks from the incubator. Perform a visual check, the culture should be slightly cloudy. Make glycerol stocks from culture if neccesary. Label centrifuge tubes with the plasmid numbers, then pour the entire contents of the flasks into the tubes (its fine if the pipette tip also goes in).
Spin tubes at 3500rcf (~6000rpm) for 10 minutes to pellet the cells. Discard supernatant by pouring off (pipette tip should come out now).
Continue by following Zymo Pure protocol.