Graduate Research Experience

Todd Bernacil DNA manipulation and Cloning. Digested pUC19 plasmid DNA with SacI restriction endonuclease at the multiple cloning site (MCS) and simultaneously digested λ phage DNA to yield an insert fragment. Ran a sample on agarose gel electrophoresis to confirm digestion. Ligated the λ fragment sticky ends with the pUC19 complementary sticky ends using ligase. Transformed fresh E. coli cells with the recombinant pUC19 plasmid with λ insert using CaCl2 coprecipitation and heat blocking. Selectively plated the competent cells on X-gal plates. Cloned clear colonies using LB broth + ampicillin. Colonies clear because lacZ gene within MCS was interrupted with foreign λ DNA preventing expression of β-galactosidase that breaks down X-gal to a bluish product. The ampicillin eliminated cells without the ampicillin resistance selectable marker on plasmid. Purified the amplified recombinant plasmids using Qiagen miniprep method. Diagnostically digested recombinant plasmid with EcoRI and HindIII restriction enzymes. Bands confirmed by gel electrophoresis. ELISA Assay Development. Developed a sandwich ELISA assay to determine optimal antigen binding using various concentrations of capturing and 1°detection Ab, and various dilutions of 2° Ab (conjugated to HRP). Also determined the concentration of unknown Ag samples by generating a standard curve of known Ag dilution concentrations. Competitive ELISA was used to compare the binding affinity of a labeled Ab to that of a competitor Ab in pursuit of binding to an anchored Ab. Concentrations of the two competitors were made. Polymerase Chain Reaction. Detected genetically modified foods with traces of plant matter using PCR and agarose gel electrophoresis. GMO master mix contained primers that recognized genetically modified DNA sequences. Plant master mix contained primers that recognized the photosystem II gene. Bands confirmed GMO while no bands didn’t. Mammalian Cell Culturing. Cultured and subcultured mouse cells (NIH3T3) and human cells (K562) using basal (DMEM; Iscove’s MDM) and serum type (BCS; FBS) media. Operated under laminar flow hood. Medium aspirated and cells released using trypsin in order to count cells using hemacytometer. Viability of cells/mL calculated and used to measure how much resuspension in fresh media to bring up cell number to late log phase. Samples of cells were then fixed in metaphase with 3:1 methanol/glacial acetic acid and dropped onto slide to break open nuclei. Chromosomes stained with Giemsa stain for karyotyping. Rest of cells cryogenically frozen using DMSO. Protein Purification and Analysis. Cloned E. coli cells in LB broth + ampicillin. Cells contained recombinant plasmids with ERK gene alongside a glutathione-S-transferase (GST) tag sequence all controlled by a lac promoter. Ampicillin resistance included. ERK gene is oncogenic; must isolate product for cancer studies. IPTG (an artificial sugar) added to cells during late log phase (measured by spectrophotometry) to induce transcription and production of ERK-GST protein complex. Froze cells with liquid nitrogen for storage. Lysed cells chemically (e.g. lysis buffer) and mechanically (e.g.

Todd Bernacil – p.2 glass dounce). Centrifuged lysates to isolate protein supernatant. Supernatant then transferred to affinity chromatography column with agarose beads that had glutathione molecules covalently anchored to them. The glutathione-S-transferase tagged onto the ERK protein recognizes and ionically binds to the glutathione substrate. Buffers applied. Elution buffer contained free glutathione molecules that competed to bind to the glutathion-S-transferase part of the ERK protein. The ERK-GST released from the agarose beads in pursuit of binding with free glutathione (elution). Small samples of these elutions applied to microtiter wells with Bradford reagent to detect color changes (protein indication). ERK sample inserted into a Slide-A-Lyzer cassette in a solution of dialysis buffer to reduce the sample volume by about 3X (more concentrated ERK protein). Prepared a BSA standard in a microtiter plate using diluted Bradford reagent. Used this standard to calculate an average concentration of several triplicate dilutions of ERK protein. Next determined purity of protein preparations collected as well as the molecular weights of the desired protein products using SDS-PAGE. Fractions collected before elution had multiple bands indicating no ERK purity. Fractions collected after elution had about two bands indicating ERK purity.