# BME100 f2014:Group27 L5

BME 100 Fall 2014 Home
People
Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
Course Logistics For Instructors
Photos
Wiki Editing Help

 Jonathan Almendras Patrick Conely Krystal Corrette Aaron Dodell Adam Samuel Yakut Umar

# PCR Lab B

20-200ul Micropipettor Challenge

1. Dial a 20 - 200 ul micropipettor to 30 ul and place the tip on it.

2. Practice pushing the plunger to the first stop (when you feel resistance).

3. Practice pushing the plunger to the second stop (when it is all the way down to the barrel).

4. Cut a strip of empty PCR tubes in half and label each set of 4 1-4.

5. Using good pipetting technique, draw up the following volumes listed in the chart below and dispense them in the appropriate PCR tube.

Figure 1: Completed chart of volumes (µl) and colors

 Tube Red Blue Green Yellow Final Color Final Volume (µl) 1 30 20 0 0 Purple 50µl 2 20 0 0 30 Orange 50µl 3 0 50 0 50 Green 100µl 4 0 0 50 50 Light Green 100µl

Questions

1. Which of the data was qualitative and which was quantitative? From the chart above, the final volume was quantitative data and the final color was qualitative data.

2. What is the difference between accuracy and precision?

Accuracy refers to how close measurements are to what they are supposed to be. Precision refers to how close they are to each other (a more relative description). For example, if the final volumes were supposed to be all 100 ul, it would be high accuracy but low precision if they were 96, 97, 103, and 106 ul. However, if they were 80, 81, 79, and 81, they would have low accuracy (not close to the target measurement of 100) but high precision.

3. In this exercise, were you accurate, precise, or both? Explain.

In this exercise, we were both accurate and precise. Our measurements aligned with the key which we compared it with, suggesting that we had pipetting technique that gave us accurate and precise volumes of each liquid.

Section 2: PCR Reaction

1. Gather all of the materials from the Lab A protocol.

2. Again, cut a strip of empty PCR tubes in half (two groups of 4).

3. Label the sides of the empty tubes as positive control, negative control, patient 1 replicate 1, patient 1 replicate 2, patient 1 replicate 3, patient 2 rep. 1, patient 2 rep. 2, and patient 2 rep. 3. We used the abbreviations PC, NC, 1-1, 1-2, 1-3, 2-1, 2-2, and 2-3.

4. Pipet 50 ul of PCR reaction mix into the positive control tube, and then transfer the positive control/primer mix into the same tube. There should be 100 ul total.

5. Repeat for each tube labeled (negative control, patient 1 (1, 2, and 3) and patient 2 (1, 2, and 3). Make sure to discard the tip after each one.

6. Tightly close the lids of the PCR reaction tubes.

7. Run the PCR when there is another group ready (all 16 slots must be filled at a time).

# PCR Lab C

Procedure

-Type of smartphone: Apple iPhone 5

Settings:

A. Flash inactivated

B. ISO pre-set to 800

C. White Balance set to Auto

D. Exposure: N/A

E. Saturation: N/A

F: Contrast: N/A

The smartphone was set in the cradle and was adjusted so that it was as upright as possible The distance (in cm) from the flourimeter was 4.2 cm. 80 microliters of SYBR Green I was added to 80 microliters of each of the calf thymus solutions. There were 6 solutions as follows:

1. 5 micrograms/mL initial concentration of 2x Calf Thymus DNA solution (2.5 ug/mL final concentration)

2. 2 ug/mL initial, 1 ug/mL final

3. 1 ug/mL initial, 0.5 ug/mL final

4. 0.5 ug/mL initial, 0.25 ug/mL final

5. 0.25 ug/mL initial, 0.125 ug/mL final

6. 0 ug/mL initial, 0 ug/mL final

Each drop had to be placed in the middle of the slide and then aligned such that the blue LED light was focused by the drop to the middle of the fiber-optic black fitting on the other side of the flourimeter.

The phone timer was then set for 3 seconds, the flap was closed, and the image was taken.

Three images were taken for each. Each droplet was then removed with a micropipettor set to 160 microliters.

# PCR Lab D

Background

PCR can be used to detect a DNA sequence which is associated with a disease, known as SNP. This is done by designing an SNP-specific primer pair.

In this lab, we used the NCBI database to find the DNA sequence and then design a SNP-specific primer pair.

Results

Part 1:

-A nucleotide is the backbone of DNA/RNA (deoxyribose sugar, base pair, and phosphate group).

- Polymorphism is the presence of multiple phenotypes in an organism.

- The variation is found in the Homo sapiens species on the chromosome 21.

- The clinical significance of this SNP is pathogenic.

- It is associated with the gremlin gene.

- Long QT syndrome and clinical long QT syndrome are linked to this SNP.

Part 2:

- KCNE2 stands for "potassium voltage-gated channel, Isk related family, member 2"

- KCNE2 encodes the potassium channel that assembles with the KCNH2 gene product to alter its function.

- An allele is 1+ genes that appear alternatively at a locus on a chromosome.

-The disease-associated allele contains the sequence CTC.

- The numerical position of the SNP is 34370656.

Part 3:

- The non-disease forward primer is (5') C A T G G T G A T G A T T G G A A T G T (3').

- The numerical position exactly 200 bases to the right of the disease SNP is 34370856.

- The non-disease reverse primer is (5') C C C T T A T C A G G G G G G A C A T T (3').

- The disease forward primer that is SNP-specific: (5') C A T G G T G A T G A T T G G A A T G T (3').

- The disease reverse primer that is SNP-specific: (5') C C C T T A T C A G G G G G G A C A T T (3').