Bio-Rad Ligation and Transformation Module - Manual (Page 38)

relatively high transformation efficiency (10 6 transformants per µg DNA)
without a requirement for a refrigerated centrifuge, commercial competent
cells, or a –70°C freezer to store the competent cells.
Note: Bio-Rad offers both chemically competent and electrocompetent
cells for purchase should your teaching goals include electroporation or
more traditional chemical transformation techniques. The commercially
available cells require storage at –70°C and have transformation efficiencies
of 10
9 /µg DNA.
Note: The Ligation and Transformation module requires microbial culturing
reagents, such as LB broth, LB agar, ampicillin, petri dishes, cell culture
tubes, and inoculation loops, which are not included in this module. You
may choose to purchase the Microbial Culturing module (catalog #166-5020EDU)
for these components.
Tasks to Perform Prior to the Transformation Laboratory
1. Prepare solid and liquid growth media at least 5 days prior to the
transformation step. The Microbial Culturing module contains all
reagents required for this activity, or alternatively the reagents may be
prepared using standard protocols. (Note: Complete instructions for
reagent preparation and streaking plates are available in the Microbial
Culturing module instruction manual.) Each student team requires:
1 LB agar plate for preparing starter colonies, 1–2 LB Amp IPTG agar
plate with final concentrations of 50 µg/ml ampicillin and 0.2 mM IPTG
for plating transformations (if performing a positive control transformation
2 plates will be required), 5 ml of LB broth for growing starter culture
and 20 ml of LB Amp broth with final concentration of 50 µg/ml ampicillin
for growing 4 minipreps. All reagents may be stored at 4°C for up to
1 month.
2. Innoculate growth media and culture cells
a. Streak an E. coli HB101 starter plate: At least 2 days prior to the
transformation, streak an E. coli culture appropriate for transforma-
tion on an LB agar plate using standard microbial techniques to
allow formation of single colonies. If using the Microbial Culturing
module, rehydrate the
E. coli HB101 vial with 250 µl of sterile water
and use 10 µl of the rehydrated bacteria to streak plates. Incubate
plate at 37°C overnight. Once colonies have grown, wrap plate in
Parafilm and store at 4°C for up to 2 weeks.
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