Growing Transformed Culture

Start your culture early! We recommend growing your culture early to make sure you get a good pink color in your cells and that it is ready to go on lab day. We usually have back-up flasks of culture that can be provided if your culture fails for some reason, but you have to pick them up and they can’t always be provided last minute. 

START first thing in the morning, on a day when you will be returning to school the next morning! This isn’t a quick process and can’t be rushed. 

1. Thaw the red-top tube of pre-transformed cells provided with your ABE reagents. It can be thawed at room temperature, or on ice if your room is particularly warm. It should NOT be warmed up (e.g. in warm water or with your hands). 
2. Add the contents of the red-top tube (500µL pre-transformed cells) to the flask of LB-Amp provided with your materials. Incubate 4-6 hours in the shaking incubator at 37°C at a shake speed that gives good movement to aerate the cells. 
3. After 4-6 hours, the culture should be cloudy with bacteria. If you hold your hand behind it you should be able to see that your hand is there, but not clearly. At this point, add the arabinose provided (1000µL). Depending on conditions getting a cloudy culture can take longer! Only add the arabinose after the cloudy stage is reached, but don’t wait too long once the culture is cloudy. You are trying to introduce the arabinose right at log-phase in the culture’s growth. 
4. Continue shaking at 37°C until the culture has been growing 24-36 hours, or a nice, ruby grapefruit color is achieved. The amount of time varies significantly depending on growing conditions, the state of the culture, etc. If you don’t have pink by 48 hours, time to give up and see if we have a back up flask for you.
5. Store your culture in the fridge (if you are using it within a few days) or freezer (up to a few months!). We recommend aliquoting it into 1mL aliquots first, so that it’s ready for your students to use. Each group will need two 1-mL aliquots for lab 6. If you don’t have a chance to pre-aliquot, thaw your culture at room temperature before use (can take an hour or longer), or in the fridge overnight.