Gene PCR

Aim: Extract and amplify our genomic UAP1 sequence from yeast DNA by Polymerase chain reaction (PCR).

Protocol: Gene PCR. We ran a 0.8% agarose gel electrophoresis to check our PCR product size.

Conclusion: We successfully amplified the UAP1 gene using PCR (Figure 1 showing a and of the size 1500bp). We can assemble our gene with a digested vector and transform it into chemically competent E. coli cells.

Vector PCR

Aim: Linearizing and amplifying our vector IBA3 by Polymerase chain reaction (PCR).

Protocol: Vector PCR. We ran a 0.8% agarose gel electrophoresis to check our PCR product size.

Conclusion: We successfully amplified IBA3 vector using PCR (Figure 1 shows a band size 3215bp). We can now digest the vector with Dpn I enzyme.

Result:

Isolation — **Figure 1:** To the left UAP1 1484bp, to the right IBA3-vector 3215bp.

Dpn I digestion

Aim: : Linearization of the IBA3 plasmid for subsequent gene assembly.

Protocol: Plasmid digestion.

Conclusion: Ready plasmid for genomic assembly.

Gene Assembly

Aim: Assemble our gene UAP1 with digested vector IBA3 and sequentially transform into competenc DHalpha E. coli cells.

Protocol: Gene assembly.

Conclusion: Ready plasmid with recombinant DNA for transformation.

Bacteria Transformation and plating

Aim: Transform our cloned vector into the competent cells, DH-alpha E. coli, heat-shock and plate them on LB plate with ampicillin.

Protocol: Transformation.

Conclusion: Four colonies grew on the plate, however, have to verify the correct assembly of the cloned DNA construct by doing a colony PCR.

Heat-shock

Mix the competent cells DH-alpha E. coli into the genes assembly and heat-shock them using thermocycler (-17 min).

Inoculation

Aim: Pick the colonies that survived the ampicillin treatment and grow them in liquid cultures to amplify our plasmid DNA.

Protocol: Batch of 5 mL E. Coli from agar plate.

Result: Turbid tubes which indicate bacterial growth.

Conclusion: Sufficient bacterial culture for subsequent experiments such as plasmid purification and protein induction.

Inoculation of liquid culture

Instead of the standard 5 mL/tube, we used 3 mL/tube
Cultures were further subcultured from established tubes to 100 mL Erlenmeyer flasks with 20 mL/flask LB w/ampicillin (100 mg/ml)

Colony PCR

Aim: To screen for genetically transformed and verify the assembly of the DNA construct.

Protocol: Colony PCR.

Result: Confirmation of expected size of amplicons.

Conclusion: Assembly and transformation of the UAP1 gene into the IBA3 vector into DHalpha competent cells were successful in 3 of the 4 colonies.

Miniprep

Aim: Isolate plasmid DNA from other DNA.

Protocol: Miniprep.

Result: Sufficient amount of plasmid DNA in 3 cultures, determined by nanodrop.

#	Concentration (ng/µL)
I1	199
I2	164
I3	99

Conclusion: Ready plasmid DNA for sequencing.

Sequencing

Aim: Prove successful insert in vector.

Protocol: 6 tubes for 3 isolated plasmid samples were prepared. 3 tubes were prepared with 5 mM forward primer, and 3 with 5 mM reverse primer. 80-100 ng/µL plasmid DNA was added to each tube, one sample in both forward and reverse tube, then topped to 10 µL total in water. Sent for sanger sequencing to Eurofins Genomics.

Result: Successful insert of plasmid in all three cultures.

Conclusion: Undisputed proof that the target gene is inserted in the bacteria.

Protein induction

Aim: Using AHTc to induce the expression of UPA1 gene.

Protocol: 10 mL bacterial culture is transferred to new flask, a dilution is prepared until 10 µL diluted AHTc is added to each.

Result: Colonies that produce the target protein.

Conclusion: Bacterial cultures in both induced and uninduced states are to be used for the analysis of protein to see if UAP1 is expressed as expected.

Cell Lysis for nuclear protein extraction

Aim: Lyse the cells without using detergent to preserve the protein function.

Protocol: Pelleted with 3 000 rcf for 8 min. Lysis buffer added to 1/10 original volume (1 mL). Lysis buffer consistent with 200 mM, 0.5 mg/mL lysozyme, and 10 mM Tris. Bead lysis for ~40 seconds. Lysate separated into new tubes, then stored on ice for processing.

Conclusion: To determine the effectiveness of the cell lysis, functional assay and sds required.