Bacterial Colonies Examination, qPCR Standard Curve, Journal Meeting

Technical Log

Name: HaoQi (Esther) Li
Date: 7/14/06 Friday
Work Location: Naval Medical Research Center, Silver Spring, MD
Title of Project: Developing and Optimizing a qPCR Assay for Borrelia lonestari

1. Objective – Why did you hope to accomplish today?

I will analyze the transformants. I plan to do a standard curve of the DNA concentration assay I’ve done a week ago.

2. Events – a. summarize the schedule of events. Procedures performed in detail, results, analysis, with tables, charts, diagrams, and scanned sketches
b. summarize communications, emails (attached), conversations, pertinent material read (annotated bibliography)+ reactions, browser used (search identifier, annotation)

Bacterial Colonies Examination:

I photos of my beautiful bacteria colonies and I’ll upload them into the computer this weekend. Since yesterday the cotton swab absorbed a lot of the bacteria solution when I was spreading them out on the 50μl half of the plate, the 50μl side had few isolated colonies, which was actually a good thing since picking out individual colonies would be easier.

However, it was not possible to pick out the individual colonies because they need to be incubated overnight, but the weekend is coming up, so we have to wait until Monday.

The edge of the plate was covered up with Parafilm so that the bacteria would have less chance of being contaminated, and then the plate was put in the 4˚C refrigerator.

qPCR Standard Curve:

For the Standard Curve

First the “Sample Type” and “FAM Std/Res” values need to be changed.

Click on “Standard 1” to view the Standard Curve, which is a line. There is a red square that indicates that one of the negative samples came out to be positive.

Indeed, one of the two dirty hood negative controls, D Neg 1, was positive, which was indicated by the brown line. In addition, 100.5 did not yield any positive results and 100 crossed the threshold before 101. Dr. Ju said that since one of the negative showed up as a positive, the results of 101, 100.5, and 100 should not be counted as valid.

The Second Derivative Test also helps to analyze the information by graphing the rate of change of the primary growth curve:

When the box rate average is changed from 0 to 3, the curves a smoother:

Journal Meeting:

Today, Dr. Ju talked about the article: “The Genome Sequence of Rickettsia felis Identifies the First Putative Conjugative Plasmid in an Obligate Intracellular Parasite”

The article talked about how special R. felis is as “the first putative conjugative plasmid identified among obligate intracellular bacteria”. A conjugative plasmid can use a pilus to transfer genetic information to another bacterium. Obligate intracellular bacteria have to stay in bacteria to survive. The authors of the article found that there were many genetic differences of R. felis from other Rickettsia strands, such as having two little plasmids, having genes extended by repetition in the main plasmid. They were able to confirm their results with many gel electrophoresis PCR tests.

Figure 1.Circular Representation of R. felis, R. conorii, and R. prowazekii Genomes

The three outer circles represent the chromosomes of R. felis, R. conorii, and R. prowazekii, respectively, with specific ORFs colored in red and nonspecific ORFs colored in black. Colinear genome fragments are highlighted by a shared background color, with their relative orientations indicated by arrows. The two inner circles represent two R. felis plasmids (pRF and pRFδ), with ORFs in the region unique to pRF colored in red.

Table 1.Comparison of R. felis and Other Published Rickettsia Genomes

3. Reflections

a. actually accomplished: I checked the bacteria culture I made yesterday and took pictures. Then I made standard curve for my DNA concentration assay last week, then I played around with the qPCR program.

b. concerns: On Monday I will be at school from 8:00am – 8:45am, but then I will rush to my lab at 11:30am to attend another meeting with other SEAP students to listen to a presentation and instructions about writing the research paper.

c. learning (workplace, science, project, yourself): I read and learned more about the qPCR data analyzing using standard curves sand second derivative curves.

4. Planning

I hope to pick out a few colonies and culture them for DNA tests.