Lab presentation

BIOCHEMISTRY & MOLECULAR BIOLOGY

Li Lan F. and Kailyn D.

Lab 1:Designing primers using Bioinfomatics

Results per qualifications: - GC% = 50-60% - Self-complementarity <=5 - Identity score = 100% - Query cover = 100%

A primer is a DNA strand that initiates replication by binding to a complementary sequence. It can be designed to target a specific sequence. DNA polymerase creates a product size in base pairs, where A pairs with T and G pairs with C. High G-C content sequences are harder to denature due to more hydrogen bonds. Query cover percentage compares a targeted sequence to a database's alignment to find a match. Percent identity indicates similarity between two sequences.

Lab 2: RNA isolation and cDNA synthesis

Objective: To isolate total RNA in the cell and to reverse transcriptase unstable mRNA sequence into a more stable DNA form called cDNA. Expectation: Purity qualification 260/280 ~ 1,8 260/230 ~ 2-2.2 Result: Total amount of RNA = 50.2 ng/uL 260/280 ratio = 1.66 260/230 ratio = 0.92 (did not meet)

Lab 3: PCR & DNA gel electrophoresis

Objective: PCR is used to amplify target DNA/RNA strands so they can be detected and separated (by size and charge) through gel electrophoresis. Expectation: Strong, visible bands of both test and sample ladders. Result: The bands for the ladder and controls are bright and easily detectable. However, the bands for the test samples are faint and difficult to observe. This could be due to impurities or low quantity of cDNA.

Control Vector: Small number of colonies were spotted, and the tube have little turbidity.

Lab 4: Bacterial Transformation

Result: Desirable results were observed in the plate and tube containing CXCL12, but not in the control samples. Two posible reasons for this discrepancies: (1) The cells did not fully transform during the previous step so they weren't able to resist against antibiotic. (2) Accidental contamination of either the cells, media, or equipment, etc so they failed to tranform.

Objective: To transform E. coli bacteria, allow it to uptake and express antibiotic resistance gene (CXCL12). Expectation: Visible colonies in plate and turbidity in tube for both control and test samples.

Test vector: There are colonies on the plate and turbidity in the tube.

Test results: Concentration = 3.6 ng/uL 260/280 ratio = 0.87 (did not meet) 260/230 ratio = 0.17 (did not meet)

Control results: Concentration = 143.3 ng/uL 260/280 ratio = 1.64 260/230 ratio = 0.52 (did not meet)

Lab 5:Plasmid Isolation

Objective: Harvest transformed plasmid DNA in bacterial cells from Lab 4 by isolation. Use nanodrop to analyze the purity of the samples. Expectation: Purity qualification Total concentration > 600 ng/uL 260/280 ~ 1,8 260/230 ~ 2-2.2 Results: Did not yield the expected outcome. Two possible reasons: 1. Excessive amount of cells were used, clogging of the filter column, DNA strands failed to pass through the flow. 2. Samples were contaminated during the extraction process (human error). Despite conducting several retries, the data did not improve, and the root cause remains undetermined.

Lab 6:Restriction Digestion Enzyme and DNA Gel Electrophoresis

Objective: To cut circular plasmid at specific sites into predictable fragments for DNA gel eletrophoresis using restriction enzymes: HINDIII (largest), NCOI, and BAM HI (smallest). Expectation: Three strong, visible bands of both test and sample ladders. Result: Two out of three bands per well can be seen upon closed observation. 3 possible causes: 1. Not enough fragments were cut to give a strong sight. 2.Contamination. 3. The samples were poorly loaded into the well.

Eureka!

Thanks!

QUESTION??

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