Woon Teck Yap
Section B, M1-3, E53-220
Meeting 2
Out-of-class Exercise 1
Write the methods section for two experiments from the Genetics/Protein
Biochemistry Module.
Methods section for Experiment V-A: P1 Transduction – Stabilization and Mapping
6 1.5 ml Eppendorf tubes were labelled “BW140” while another 6 were labelled “C600”.
The 6 sets of Eppendorf tubes, each consisting of 1 “BW140” tube and 1 “C600” tube,
were then labelled with “O3W1”, “L3W5”, “N3W2”, “O3W2”, “BK1” and “No P1
lysate”, respectively.
1 ml aliquots of overnight cultures of BW140 or C600, in LB broth + 5 mM CaCl2, were
then pipetted into the appropriately labelled tubes and centrifuged at 6000 rpm for 2
minutes. The resulting supernatants were then poured off and the pelleted cells were then
resuspended in 0.3 ml MC medium (10mM MgSO4, 5mM CaCl2). 10 μl of the P1 lysate
obtained from the O3W1 mutant was then added to the 2 Eppendorf tubes labelled with
“O3W1”. The process was then repeated 4 more times for P1 lysates obtained from the
L3W5, N3W2, O3W2 and BK1 mutants. No P1 lysate was added to the tubes that had
been labelled “No P1 lysate”. The mixtures of bacterial cells and P1 lysates were shaken
gently to induce mixing and then set aside on the bench to incubate for 30 minutes at
24.5C.
100 μl of 1M sodium citrate was subsequently added to each Eppendorf tube. The
resulting mixtures were then gently swirled and transferred to sterile culture tubes. 1 ml
LB medium was added to each culture tube which were then placed on a roller drum and
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Woon Teck Yap
Section B, M1-3, E53-220
Meeting 2
Out-of-class Exercise 1
incubated at 37C for 1 hour. Following the incubation, the cultures were transferred to
Eppendorf tubes and centrifuged at 6000 rpm for 2 minutes. The supernatants were then
decanted and each resultant pellet was resuspended in 50 μl of LB broth + 0.1M sodium
citrate. Each suspension was then plated onto an appropriately labelled LB Kan plate and
incubated overnight at 37C.
The bottom of the remaining LB Kan plate was then marked with 5 evenly-spaced dots,
each of which was labelled “O3W1”, “L3W5”, “N3W2”, “O3W2” and “BK1”,
respectively. After 5 μl of the respective P1 lysate was spotted onto the appropriate dot,
the plate was then set aside and allowed to dry undisturbed.
Methods section for Experiment I-B: Streaking Bacteria on Agar Plates
Each of the 4 plates (Mac Ara Kan, Mac Lac Kan, LB Kan and LB Ara X-gal Kan) was
divided up into 4 equal-sized sectors by drawing 2 mutually orthogonal lines on the
bottom side of each plate. The 4 sectors on each plate were then labelled “BK1”, “EJ1”,
“H24” and “JET2”. A sterile toothpick was used to pick a single colony of the BK1
strain from the provided plate and inoculate it onto BK1 sectors on all 4 plates. The
process was then repeated for the EJ1, H24 and JET2 strains. Each inoculation was then
streaked out using sterile sticks. All 4 plates were subsequently incubated overnight at
37C in the warm room.
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