Basic Molecular Techniques

Introduction:

The purpose of this lab is to introduce students to basic laboratory techniques used in molecular genetics. This lab covers the use of micropipets and the asceptic technique for the transfer and culture of bacteria.

Materials:

Part 1 and 2:

Part 3:

5 - 1.5 mL tubes with 1 mL each colored solution E.coli stab culture or plate

Sterile LB medium E.coli/pAMP stab culture or plate

4 sterile culture tubes 2 - LB plates

0.5 to 10 µL digital micropipet and tips 2 - LB-AMP Culture plates (LB+Ampicillin)

100 to 1000 µL digital micropipette and tips Inoculating loop

10 mL pipet Bunsen burner

50 mL conical tube Permanent marker

15 mL culture tube

1.5 mL reaction tubes

500 mL beaker

Bunsen burner

Permanent marker

Procedure:

Part 1A:

Three 1.5 mL reaction tubes were labeled A, B, and C. A matrix was written down to be used as a checklist as solutions were added. 4 µL of solution I were delivered to each tube using the 0.5 - 10 µL pipet. A fresh tip was used to add the approximate volume of Solution II to a clean spot on each reaction tube. Another fresh tip was used to add 1 µL of Solution II to tubes A and C. Finally, another fresh tip was used to add 1 µL of Solution IV to tubes B and C. The tops of the tubes were closed and placed in a balanced configuration in the microfuge rotor. The tubes were spun for a 1-2 second pulse in the microfuge. The solutions were withdrawn from each tube to check the accuracy of the pipetting.

Part 1B:

Two reaction tubes were labeled E and F, and the above procedure was repeated to determine the accuracy of the pipetting using a 1000 µL pipet.

Part II:

The lab bench was cleared and decontaminated and was set with a container of sterile LB (Luria Broth) medium, a set of 4 culture tubes, sterile 10 mL pipets, pipet aid, and Bunsen burner. The culture tubes were labeled with the group name's initials. The Bunsen burner was lighted and use to minimize contamination. The pipet aide was set to 5 mL to expel contaminated air and prepared for the vacuum to withdraw fluid. A sterile 10 mL pipet was then inserted into the pipet aide and was used to withdraw 5 mL of medium after the flame was used to quickly pass over the container. The top was reflamed and placed back into the rack.

Part IIIA:

The bottom of each agar plate was marked with our team name and what type of LB plate it was. They were marked LB plate A, LB Plate C, LB-AMP plate B, and LB-AMP D. The loop was held like a pencil and sterilized in the Bunsen flame until it was glowing red and was then passed to the lower 2/3 of the shaft through the flame. The loop was cooled for 10 seconds, was used to make several streaks across the top of the plate. The loop was reflamed and cooled by stabbing it into the side of the agar. The cooled looped was used to draw through the first streak and continued to streak