BME100 s2014:T Group11 L4
Lab 4 Write-Up
OUR TEAM
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Initial Machine Testing
The Original Design
The open PCR machine that will be used in this experiment is shown below. The machine will be used to heat and cool the DNA samples in the experiment in order to amplify the targeted DNA sample. To do this the machine consists of a heating lid, PCR block, LCD screen, heat sync/fan and a power supply. The heating lid is used by the machine to heat the PCR block, which holds the DNA sample. If this part does not work properly the samples will not be heated to the necessary temperatures, thus the PCR reaction will not take place. The LCD screen displays the temperature of the heating lid and the active step of the experiment. The thermal conductor and fan both heat and cool the machine. If this part of the machine is not working correctly the PCR block, holding the PCR samples would not cool or heat properly. The power supply, like the name suggest is how the machine and it's parts get their power.
Testing the Machine
The PCR machine is a complex machine and in order to make sure it was working properly it was necessary to test the connections of the machine and ensure that all the parts were not only working properly on their own but also that the parts were communicating properly with each other. To do this the LCD display was disconnected from the power supply which caused the LCD screen to be turned off. This was reconnected and then the temperature sensor was disconnected from the PCR block. This resulted in the temperature sensor to display both inaccurate and extreme temperatures. After everything was reconnected the PCR machine was run though a thirty five cycle test run. During this test run the machine was unable to complete one cycle. The cycle was inhibited because the machine could not cool the PCR block. Once the temperature of the block was raised to 95 degrees Celsius the machine was unable to bring the temperature of the block back down to any temperature below 80 degrees Celsius. Due to the machine's inability to cool it was assumed that there was an error in the function of the heat sync/fan. The fan was unable to draw in enough air to cool the machine resulting in the improper function or failure of the machine.
Labeled Thermocycler:
Protocols
PCR Machine Settings
Initial Step:
Temperature:95°C
Time: 180 seconds
Number of Cycles: 35
Step:
Temperature: 95°C
Time: 30 seconds
Step:
Temperature: 57°C
Time: 30 seconds
Step:
Temperature: 72°C
Time: 30 seconds
Final Hold:
Temperature: 4°C
DNA Sample Set-up
Two different patient's DNA samples were tested along with a positive and negative control. Patient 1's ID number is 30337 and Patient 2's ID number is 57707. The samples for Patient 1 were labeled P11, P12, and P13. Patient 2's samples were labeled P21, P22, and P23. The positive control was labeled PCD and the negative control was labeled PCN. The table below shows how the reaction tubes were arranged and labeled:
Tube Labels
| PCD | P11 | P12 | P13 |
| PCN | P21 | P22 | P23 |
DNA Sample Procedure
1. 8 empty PCR reaction tubes were obtained and labeled according to the above diagram.
2. 50μL of PCR reaction mix was placed in each of the tubes (this reaction mix was the same for all the DNA samples).
3. 50μL of DNA/primer mix was added to each of the tubes. This mix was different for each of the tubes based upon whether the tube was a control or patient sample. The corresponding sample was added to the corresponding tube.
4. The reaction tubes were placed into the PCR block of the PCR machine.
5. The PCR machine was started and temperature cycles were completed.
Components of the PCR Reaction Mix
The PCR reaction mix is a mixture that contains TAQ polymerase, MgCl2 and the Deoxyribonucleotides (dNTPs). This is the reaction mix that is the same for all the reaction tubes. The TAQ polymerase is the protein that attaches to the single stranded DNA and then adds the dNTP's to this template DNA strand. the Deoxyribonucleotides are the individual building blocks of the new strand of DNA and are added by the polymerase to pair with the template strand of DNA.
Components of the DNA/Primer Mix
The DNA/Primer mix is composed of the template DNA and the primers for the desired DNA sequence. This is the reaction mix that varies for all the samples. The template DNA in this particular experiment comes from the patients and the supplied positive control and negative control. The primers are specific, man made sequences that attach to the template DNA bordering the DNA sequence that needs to be amplified.
Research and Development
In this experiment there were 4 main components involved. There was the Template DNA which was just the original DNA used for replication. Then there were the primers which attach to the Template DNA, starting point replication. Taq Polymerase allows for addition of new nucleotides to the Template DNA. Finally there was Deoxyribonucleotides (dTNP’s) these were the floating nucleotides that added to the primers during replication. These dTNP’s are what constitutes the replicated strand of DNA.
For this experiment there were 5 steps for the thermocycling. The first step was place the samples at 95°C for 3 minutes. At this temperature the double stranded DNA is denatured, allowing for single stranded DNA and thus replication. Then the machine goes to 57°C for 30 seconds. This is the Anneal phase where, at this temperature, the primers attach to the denatured, single stranded template DNA. Following this, the machine cycles to 72°C for 30 seconds, called the Extend Phase. During this time the polymerase attaches dTNP’s to the template DNA (making it double stranded again). The temperature is then held at this themperature for 3 minutes so the DNA can replicate. Finally, the machine goes to 4°C to stop the PCR reaction.
Adenine—Pairs with—Thymine
Thymine—Pairs with—Adenine
Cytosine—Pairs with—Guanine
Guanine—Pairs with—Cytosine
