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Todd Bernacil

DNA manipulation and Cloning. Digested pUC19 plasmid DNA with SacI restrictionendonuclease at the multiple cloning site (MCS) and simultaneously digested phage

DNA to yield an insert fragment. Ran a sample on agarose gel electrophoresis to confirm

digestion. Ligated the fragment sticky ends with the pUC19 complementary sticky endsusing ligase. Transformed freshE. coli cells with the recombinant pUC19 plasmid with

insert using CaCl2 coprecipitation and heat blocking. Selectively plated the competent

cells on X-gal plates. Cloned clear colonies using LB broth + ampicillin. Colonies clearbecause lacZ gene within MCS was interrupted with foreign DNA preventing

expression of -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. Diagnosticallydigested recombinant plasmid with EcoRI and HindIII restriction enzymes. Bands

confirmed by gel electrophoresis.

ELISA Assay Development. Developed a sandwich ELISA assay to determine optimalantigen binding using various concentrations of capturing and 1detection Ab, and

various dilutions of 2 Ab (conjugated to HRP). Also determined the concentration ofunknown 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 primersthat recognized genetically modified DNA sequences. Plant master mix contained

primers that recognized the photosystem II gene. Bands confirmed GMO while no bandsdidnt.

Mammalian Cell Culturing. Cultured and subcultured mouse cells (NIH3T3) and human

cells (K562) using basal (DMEM; Iscoves 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 anddropped onto slide to break open nuclei. Chromosomes stained with Giemsa stain for

karyotyping. Rest of cells cryogenically frozen using DMSO.

Protein Purification and Analysis. ClonedE. 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. ERKgene 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.

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glass dounce). Centrifuged lysates to isolate protein supernatant. Supernatant thentransferred 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 theseelutions 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 thisstandard 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 hadabout two bands indicating ERK purity.