Molecular Techniques

Introduction

For biological experiments, there are a number of specific techniques needed to be proficient in for one to perform the investigation well. This experiment is meant to practice these techniques, and has exercises in micro-pipetting, aseptic technique, and plate streaking. These three applications of molecular techniques are invaluable skills to have in the lab and can be carried out in practice to gain experience and also provide valid feedback.

Method

The first exercise, pipet techniques, involves the use of multiple variations of micropipettes devices and their respective tip. After going over proper operation of the device, these micropipettes were used with 5 labeled colored water solutions to visualize the movement of miniscule amounts of liquid (microliter scale) into small plastic vials. The stock solutions (I – IV) were used to make 3 samples (A-C) and another stock solution, V was used to make samples E and F as follows.

Stock (µL) A (µL) B (µL) C (µL)
I 4 4 4
II 5 5 4
III 1 - 1
IV - 1 1
Total 10 10 10

Stock (µL) E (µL) F (µL)
V 100 150
V 200 250
V 150 350
V 550 250
Total 1000 1000

After preparing the samples, they were placed properly balanced into a centrifuge (just to ensure all liquid together on bottom) and a micropipette dialed to the total volume was used to confirm that the proper amount was in each. The second exercise involved the used of aseptic technique with standard graduated pipettes. A container of sterile Luria Broth (LB) medium and an empty culture tube were used to illustrate aseptic technique via a sterile 10mL pipette. The pipette was to be used quickly after being removed from the packaging, and the ends of each tube were flamed with a provided Bunsen burner to ensure a sterile environment as the LB was moved into the culture tube. The tube was then set aside labeled to be observed for colony growth next lab section. The third and final exercise was to perform proper streaking techniques on a culture plate so that isolated colonies could be obtained. Two strains of E. Coli, one normal and one with the pAMP plasmid, were used with two culture plates, one normal LB and the other LB-amp (with ampicillin). After marking the plates, an inoculating loop was flamed through the burner and cooled for 5 seconds before flaming the culture vial, and dipping the loop into it and streaking a quarter of the plate. The loop was then put into the flame again, cooled, and run through the previously streaked section and then used to streak another quadrant of the plate, and repeated until four quadrants had been streaked. This was repeated for each E. Coli culture on both kinds of plates, for a total of 4 plates. They were then labeled and set aside for observations on the next lab period.

Results

Exercise 1 – End step micropipetting resulted in no leftover liquid in the vial.

Exercise 2 – The culture tubes were only slightly cloudy, or clear.

Exercise 3 – The plates had growth as follows: (+ indicates growth, - indicates none)

Culture LB Plates LB-Amp Plates
E. Coli A. + B. -
E. Coli pAMP