BME100 s2014:T Group16 L4

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BME 100 Spring 2014 Home
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Lab Write-Up 1 | Lab Write-Up 2 | Lab Write-Up 3
Lab Write-Up 4 | Lab Write-Up 5 | Lab Write-Up 6
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Contents

OUR TEAM

Name: Angie Chan
Name: Angie Chan
Name: Jossel Nkunzi
Name: Jossel Nkunzi
Name: Ryan Hess
Name: Ryan Hess
Name: Alireza Momeni
Name: Alireza Momeni
Name: Jospeh Sidman
Name: Jospeh Sidman

LAB 1 WRITE-UP

Initial Machine Testing

The Original Design
image:Pcr16.JPG


The device displayed above is called a "PCR Machine" or Polymerase Chain Reaction. Its main function is to replicate a sample of DNA. By putting in a sample of DNA and the right ingredients, this machine will heat it and cool it to the right temperatures to replicate the DNA repeatedly. The ingredients needed include two polymers, Taq Polymerase, complimentary base pairs. The sample of DNA will be mixed with these ingredients in a test tube that will be placed in the machine. Once the heating starts, the DNA strand separates and as the machine cools, the primers attach to specific regions of the separated DNA strands. Then the polymerase attaches to the primers and completes the replication of the stands. This process will repeat over and over for many cycles until the wanted amount of DNA.

Experimenting With the Connections

When we unplugged the LSD Screen(part 3) from the motherboard(part 6), there was no information displayed on the screen.

When we unplugged the white wire that connects the motherboard (part 6) to heat block(part 2)which allows for correct temperature measurements, the machine was unable to record the accurate temperature.

Test Run

The initial test run was conducted on March 20, 2014. After downloading the software, we observed the internal and external features of the PCR machine. We conducted a test run in which we used empty tubes and set the temperatures as prescribed in the lab manual. The initial run was completely unsuccessful. The machine had only heated up to a temperature of 62 degrees and no cycles were initiated. After about 10 minutes, we stopped the experiment due to suspicion of smoke and smell. Upon opening, we found that the tubes had melted. The experiment is marked as a failure as the machine had failed to complete any cycles or reach the desired temperatures, causing the tubes to overheat and melt. One possible error worth noting, was the possibility of the top screw being over-tightened adding pressure on the inner plate and resulting in the melting of the test tubes.





Protocols

Thermal Cycler Program

The program was set as follows: -HEATED LID: 100°C -INITIAL STEP: 95°C for 2 minutes -NUMBER OF CYCLES: 35 -Denature at 95°C for 30 seconds -Anneal at 57°C for 30 seconds -Extend at 72°C for 30 seconds -FINAL STEP: 72°C for 2 minutes -FINAL HOLD: 4°C


Step 1. 1 Cycle, 95 °C for 120 seconds Step 2. 35 Cycles, 95 °C for 30 seconds, 57 °C for 30 seconds, 72 °C for 30 seconds Step 3. 1 Cycle, 72 °C for 120 seconds Step 4. Final temperature is 4°C Separation of two strands of DNA is occurred at 95 °C, at 57 °C the primers attach to their respective sites on the single stranded DNA, and finally DNA polymerase becomes active at 72°C and DNA’s are replicated .This circle repeats 35 times. By cooling the DNA’s to 4°C the PCR reaction is stopped.


Image:thermoProgram.jpg

DNA Sample Set-up

Positive control A1 Patient 54764 A2 Patient 54764 A3 Patient 54764
Negative control B1 Patient 51843 B2 Patient 51843 B3 Patient 51843


DNA Sample Set-up Procedure

1. Preparing required materials that are 8 empty PCR tubes ,PCR reaction mix ,template DNA, positive and negative control and 200 μL micropipettor

2. Labeling the 8 tubes and placing them in two different rows ( four in each row)

3. transferring 50 μL of the given PCR reaction mix into the eight labeled tubes by using the micropipette

4. Adding the negative and the positive controls to the first two of the tubes which are place in different rows.

5. Transferring DNA of patient 1 to the next three tubes(A2,A3,A4) of the first row which its first tube contains positive control , and DNA of patient 2 to the next three tubes (B2,B3,B4) of second row which its first tube contains negative control

6. Close the lids tightly on your PCR reaction tubes

7. Placing tubes in PCR machine


PCR Reaction Mix PCR Reaction Mix Taq DNA polymerase MgCl2 dNTP’s

DNA/ primer mix Patient’s DNA Forward primer and reverse primer




Research and Development

PCR - The Underlying Technology

Collecting data for Pipette Practicing: We observed both the final color and volume in micro-liters and recorded that as data. The color observations are our qualitative data and the measurements of the volumes are our quantitative data. In engineering, accuracy is defined as the closeness of measurements of a quantity to that of the actual value (or expected value or outcome) and precision describes the consistency of the results, regardless of its accuracy. Accuracy can happen, also, regardless of precision, as we may achieve the desired data, but not achieve it consistently. This case is true for our experiment with the pipette practicing, as our results were inconsistent.



Section 3: Disease SNP-Specific Primer Design: A nucleotide is a sub-unit of DNA. They are composed of a base, sugar, and phosphate group. A single nucleotide change during the copying segment is called polymorphism, so SNP is single-nucleotide polymorphism. To understand this, we observed a homo-sapien fragment of DNA processed with polymorphism. The chromosome variation obvserved was located at 6:149721690. The listed "Clinical significance" of this SNP is 'other', and the genes associated with it is SUM04 (stands for small ubiquitin-like modifier 4, which encodes small ubiquitin-related modifiers that are attached to proteins and control the target proteins' sub-cellular localization, stability or activity.) and TAB2. Diseases linked with this particular SNP are Type 1 diabetes, VKH syndrome, Rheumatoid arthritis.


An allele is an alternate form of a gene. The disease-associated allele contains the bases ATG, which was present in our observed DNA. The numerical position of the SNP is 149721690. Forward primer of the DNA sequence at that location is as follows: 5' TGCACGTCCATTGCGATATG. The other primer is exactly 200 bases to the right of the first primer and that is located at 149721890; its reverse primer is 5' AGTTTTCTAATTGAGAATGC.


If you use the disease specific primer, they will only attach to the strand of DNA that is paired with the primer. When using it with a non-disease allele, the primer cannot attach and recreate strands. Therefore, no results will show, since nothing will be recreated for us to observe.



(BONUS points: Use a program like Powerpoint, Word, Illustrator, Microsoft Paint, etc. to illustrate how primers bind to the cancer DNA template, and how Taq polymerases amplify the DNA. Screen-captures from the PCR video/ tutorial might be useful. Be sure to credit the sources if you borrow images.)





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