BME100 f2013:W900 Group13 L4: Difference between revisions
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| [[Image:allisondaisy.jpg|100px|thumb|Name: Allison Marley<br>Role(s): Research And Development]] | | [[Image:allisondaisy.jpg|100px|thumb|Name: Allison Marley<br>Role(s): Research And Development]] | ||
| [[Image: | | [[Image:MrAngell.jpg|100px|thumb|Name: Tyler Angell<br>Role(s): Initial Machine Testing]] | ||
| [[Image: | | [[Image:CoryThing.jpg|100px|thumb|Name: Cory Riecken<br>Role(s): Initial Machine Testing]] | ||
| [[Image: | | [[Image:MsReem.jpg|100px|thumb|Name: Reem Gerais<br>Role(s): Protocol Planning Specialist]] | ||
| [[Image:MrAndrew.jpg|100px|thumb|Name: Andrew Luc<br>Role(s): Protocol Planning Specialist]] | | [[Image:MrAndrew.jpg|100px|thumb|Name: Andrew Luc<br>Role(s): Protocol Planning Specialist]] | ||
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'''The Original Design'''<br> | '''The Original Design'''<br> | ||
[[Image:allisonopenpcr.jpg]][[Image:openPCR.jpg]]<br> | |||
The OpenPCR machine runs PCR reactions and can be monitored by a computer to which the machine can be connected with a USB 2.0 cable. The machine has a heated lid which prevents condensation on the caps of the PCR tubes. If this part did not work, water would condensate on the cap and cause the salt concentration to be too high for the polymerase to function properly. The heated lid is situated over the 16 tube PCR block, which holds and heats the samples in the PCR tubes. If the block did not work, the samples would not be heated to the necessary temperatures and there would not be a PCR reaction. Information about the reaction is displayed on the screen located on top of the box. Inside the box there are the heating components for the PCR block. These heating components go to a heat sink which leads into an air filter which a fan moves air through so as to keep the temperature within the box from getting too hot and the unit from overheating. The wiring for the PCR block, the cooling system, and the display are all hooked to a motherboard situated at the bottom of the box. The motherboard controls the temperature through a connection to the PCR block and runs the OpenPCR machine. <br> | |||
Images from:<br> | |||
http://openwetware.org/wiki/BME103:T130_Group_10 <br> | |||
http://www.forbes.com/sites/tedgreenwald/2011/12/31/dna-sequencing-for-fun-and-profit-a-low-cost-platform-for-garage-biotech/<br> | |||
'''Experimenting With the Connections'''<br> | '''Experimenting With the Connections'''<br> | ||
When we unplugged (part 3) from | When we unplugged (part 3) from either end, the machine's screen turned off. When we plugged the cord back in, the screen turned back on. We believe this part supplies power to the screen. | ||
When we unplugged the white wire that connects (part 6) to (part 2), the machine showed no noticeable change. However, we believe that the temperature controls will not work when the white wire is disconnected. | When we unplugged the white wire that connects (part 6) to (part 2), the machine showed no noticeable change. However, we believe that the temperature controls for the thermocycler will not work when the white wire is disconnected. | ||
'''Test Run''' | '''Test Run''' | ||
Test Date: 10/23/13 | |||
Experience: The Open PCR's time functioned properly, but the component that keeps track of the cycles did not. The unit indicated that it was on the first cycle of thirty-five throughout the duration of the PCR reaction. | |||
(Write the date you first tested Open PCR and your experience(s) with the machine)<br> | (Write the date you first tested Open PCR and your experience(s) with the machine)<br> | ||
<br><br> | <br><br> | ||
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'''DNA Sample Set-up'''<br> | '''DNA Sample Set-up'''<br> | ||
<small>Table #1</small> | |||
{| {{table}} | {| {{table}} | ||
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ID:22707 | ID:22707 | ||
|} | |} | ||
'''DNA Sample Set-up Procedure''' | '''DNA Sample Set-up Procedure''' | ||
1. Step 1: Label each tube with its respective contents to prevent misdiagnosing patients. (Refer to | 1. Step 1: Label each tube with its respective contents to prevent misdiagnosing patients. (Refer to Table #1 for labelling) <br> | ||
2. Step 2: Use pipettes to dispense 50 μL of the PCR reaction mixes into each of the 8 tubes.<br> | 2. Step 2: Use pipettes to dispense 50 μL of the PCR reaction mixes into each of the 8 tubes.<br> | ||
3. Step 3: Using different disposable pipette tips to dispense 50 μL of each DNA sample into their respective tubes<br> | 3. Step 3: Using different disposable pipette tips to dispense 50 μL of each DNA sample into their respective tubes<br> | ||
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'''PCR - The Underlying Technology'''<br> | '''PCR - The Underlying Technology'''<br> | ||
'''''Function of Components in PCR reaction''''' <br> | |||
'' | |||
The template DNA is the strand of DNA that is used to create a complementary strand. This DNA strand is used to double to amount of DNA per cycle. After the DNA is placed in the test tube, the primers are added. Since the primers have any sequence of nucleotides desired, they can then attach to sites on the DNA strands that are at either end of the segment you want to copy. The primers are powerful for copying specific DNA sequences since there is no chance they will target the wrong site. After the primers are added, the Taq DNA polymerase is added to the test tube. Taq polymerase functions to copy a cell's DNA before it divides into two seperate strands. When Taq polymerase reaches a primer that's base paired with a longer piece of DNA, it attaches itself and starts adding primers. MgCl2 is required fro Taq polymerase to function effectively. When th enzyme polymerase binds to the DNA strand, it requires Mg+ ions with OH- groups to remove a hydrogen proton from the deoxyribose of th enucleotide in order to add the next nucleotide. The deoxyribonucleotides (dNTP's) are then added. The dNTP's are single units of the bases A,T,G,and C. These bases are then used as genetic building blocks to create billions of DNA copies.Adenine (A) will pair with Thymine (T) ans Guanine (G) will pair with Cytosine (C). <br> | The template DNA is the strand of DNA that is used to create a complementary strand. This DNA strand is used to double to amount of DNA per cycle. After the DNA is placed in the test tube, the primers are added. Since the primers have any sequence of nucleotides desired, they can then attach to sites on the DNA strands that are at either end of the segment you want to copy. The primers are powerful for copying specific DNA sequences since there is no chance they will target the wrong site. After the primers are added, the Taq DNA polymerase is added to the test tube. Taq polymerase functions to copy a cell's DNA before it divides into two seperate strands. When Taq polymerase reaches a primer that's base paired with a longer piece of DNA, it attaches itself and starts adding primers. MgCl2 is required fro Taq polymerase to function effectively. When th enzyme polymerase binds to the DNA strand, it requires Mg+ ions with OH- groups to remove a hydrogen proton from the deoxyribose of th enucleotide in order to add the next nucleotide. The deoxyribonucleotides (dNTP's) are then added. The dNTP's are single units of the bases A,T,G,and C. These bases are then used as genetic building blocks to create billions of DNA copies.Adenine (A) will pair with Thymine (T) ans Guanine (G) will pair with Cytosine (C). <br> | ||
''' | '''''Thermal Cycling'''''<br> | ||
The initial step of PCR occers at 95 degrees celcius for 3 minutes. In this stage the initial denaturation occurs, with an additional 30 seconds the DNA double helix separates, creating two-single stranded DNA molecules. Next the temperature is lowered to 57 degrees celcius for a period of 30 seconds. During the annealing stage, single stranded DNA molecules naturally attempt to pair up but there are much more primer sequences than DNA. The primers crowd their way in and lock onto their target before the strands have a chance to rejoin. Next, at 72 degrees celcius for 30 seconds, the DNA polymerase is activated and locates a primer attached to a single DNA strand. It begins to add complementary nucleotides onto the strand. A final step at 72 degrees celcius for 3 minutes occurs where a final extension period occurs during this time. A final hold then takes place at 4 degrees celcius during this time the cylcle refrigerates the DNA for a final hold. This minimizes any polymerase activity that might occur at higher temperatures. | The initial step of PCR occers at 95 degrees celcius for 3 minutes. In this stage the initial denaturation occurs, with an additional 30 seconds the DNA double helix separates, creating two-single stranded DNA molecules. Next the temperature is lowered to 57 degrees celcius for a period of 30 seconds. During the annealing stage, single stranded DNA molecules naturally attempt to pair up but there are much more primer sequences than DNA. The primers crowd their way in and lock onto their target before the strands have a chance to rejoin. Next, at 72 degrees celcius for 30 seconds, the DNA polymerase is activated and locates a primer attached to a single DNA strand. It begins to add complementary nucleotides onto the strand. A final step at 72 degrees celcius for 3 minutes occurs where a final extension period occurs during this time. A final hold then takes place at 4 degrees celcius during this time the cylcle refrigerates the DNA for a final hold. This minimizes any polymerase activity that might occur at higher temperatures. | ||
Latest revision as of 16:05, 29 October 2013
BME 100 Fall 2013 | 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 | |||||||||||||
OUR TEAMLAB 1 WRITE-UPInitial Machine TestingThe Original Design Experimenting With the Connections When we unplugged (part 3) from either end, the machine's screen turned off. When we plugged the cord back in, the screen turned back on. We believe this part supplies power to the screen. When we unplugged the white wire that connects (part 6) to (part 2), the machine showed no noticeable change. However, we believe that the temperature controls for the thermocycler will not work when the white wire is disconnected. Test Run
Test Date: 10/23/13
Experience: The Open PCR's time functioned properly, but the component that keeps track of the cycles did not. The unit indicated that it was on the first cycle of thirty-five throughout the duration of the PCR reaction.
(Write the date you first tested Open PCR and your experience(s) with the machine)
ProtocolsThermal Cycler Program 1. Begins with the initial denaturation step which consists of one cycle at 95C for three minutes 2. Moves next to the denaturation step which consists of 35 cycles at 95C for 30 seconds, the anneal step which includes 57C for 30 seconds, and the extended step including 72C for 30 seconds for each cycle. 3. The Final extension step occurs at 72C for 3 minutes 4. The refrigeration of the DNA occurs during the final hold at 4C
DNA Sample Set-up Procedure 1. Step 1: Label each tube with its respective contents to prevent misdiagnosing patients. (Refer to Table #1 for labelling)
PCR Reaction Mix •Taw DNA Polymerase •MgCl2 •dNTP
•A Forward Primer •A Reverse Primer Six of the eight tubes will contain unique DNA samples from two different patients. The remaining two tubes will contain control templates with and without cancerous DNA: the Positive Control Tube will contain the cancerous DNA and the Negative will not.
Research and DevelopmentPCR - The Underlying Technology
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