Bio-Rad Ligation and Transformation Module User Manual

Page 50

Appendix A
Inoculating a Bacterial Colony for Plasmid
Miniprep

Once the plasmid has been introduced into living bacterial cells and the
cells have grown and divided on selective medium, the next step in the
experiment is to prepare a miniprep of the plasmid DNA for sequencing or
further experiments. The three steps are: 1) growing cells in liquid culture;
2) purifying the plasmid DNA from the culture; and 3) performing a
restriction digest on the purified DNA to determine if the DNA insert in the
plasmid is the expected size. To start the liquid culture, cells from an isolated
bacterial colony are placed in selective medium (nutrient broth with
antibiotic). It is important to choose an isolated single colony from the
plate, so that the liquid culture will contain cells that all have the same
plasmid. If cells from more than one colony are used for inoculation, the
miniprep may contain multiple plasmids and the DNA may not be used for
sequencing or further experiments.

Before proceeding to the miniprep protocol, grow transformed bacterial
colonies in liquid cultures using the following instructions:

1. Prepare 25 ml of LB Amp broth (if not already prepared; refer to

instructions in the Microbial Culturing module, catalog #166-5020EDU).

2. Label four 15 ml culture tubes with your initials and “pJet”, the name of

your gene, and #1 through #4.

3. Using sterile technique, pipet 5 ml of LB Amp broth into each of the

four 15 ml culture tubes.

4. One day prior to the next laboratory session, use a sterile loop or a

sterile pipet tip to pick a single colony from the LB Amp IPTG agar plate
containing the plated bacteria transformed with your ligation reaction.
Inoculate an LB Amp culture tube with the colony. Repeat for a total of
four miniprep cultures from four individual colonies.

Note: Occasionally satellite colonies may grow using this ligation method.
Pick the large individual colonies, not the tiny colonies surrounding larger
colonies. Be sure that a single colony is picked, or you may isolate multiple
plasmids from your miniprep and these cannot be sequenced.

Place the miniprep cultures to grow in a shaking incubator or water
bath at 37°C overnight.