BME103:T130 Group 13: Difference between revisions
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<br>13) Record the following: type of smartphone used, the distance from the base of the smartphone cradle to the measurement device (in cm), and attach one image for each position of the drop (5 images total). | <br>13) Record the following: type of smartphone used, the distance from the base of the smartphone cradle to the measurement device (in cm), and attach one image for each position of the drop (5 images total). | ||
After assembling the fluorimeter, you can now | After assembling the fluorimeter, you can now determine if you've amplified the targeted DNA in your PCR experiment. Using the Fluorimeter, you can calculate the relative amount of DNA through fluorescence, which is generated by excitation and emission wavelengths. In order to detect fluorescence when dsDNA is present, you'll be using SYBR Green I because it's more safer compared to other dyes. With that being said, gloves '''must''' be worn when handling any liquid containing SYBR Green I. The fluorimeter itself is a very simple machine because it uses optical caustic, a special type of optics that completely removes the need for lasers, mirrors, or lenses. Also the flourimeter is battery-powered, lightweight and portable; this allows every student to have one of these at their lab table. Following the steps below, you can easily learn how to dye your amplified DNA. | ||
<br>1)On your lab table, you'll find eight samples from the Open PCR, 1 DNA sample(calf thymus standard at 2 micrograms/mL), and water from the scintillation vial (white cap) to analyze. | |||
<br>2)With a permanent marker, label your Eppendorf tubes and number your pipettes (on the bulb part) so that no cross-contamination will occurs. At the end, you should have 10 Eppendorf tubes and 10 pipettes clearly labeled (see Table 3). '''REMINDER:''' Use only 1 transfer pipette per sample!!! | |||
<br>3)Transfer each sample separately ('''using 1 pipette per sample''') into an Eppendorf tube containing 400 mL of buffer. Clearly label this tube with the number of the sample and make sure to get all of the sample into the Eppendorf tube. ONLY use the sample number transfer pipette to place a drop onto the fluorescence measuring machine, and then discard it. | |||
<br>4)Take Eppendorf tube labelled SYBR Green I and using the specially labeled pipette, place 2 drops on the first two centered drops. | |||
<br>5)Now take your diluted sample and place 2 drops on top of the SYBR Green I solution drops. | |||
<br>6)Let the smartphone operator take as many pictures as needed. | |||
<br>7)Now you may either rerun the sample again or discard the sample pipette, but keep the SYBR Green I labelled pipette. Also you can only run 5 samples per glass slide. If more are needed ask your lab TA or professor. | |||
<br>8)Before completing the lab, run the water from the scintillation vial as a BLANK using the same procedure. | |||
<br>'''Transferring the images from your smartphone to the laptop that has ImageJ''' | <br>'''Transferring the images from your smartphone to the laptop that has ImageJ''' |
Revision as of 21:34, 14 November 2012
BME 103 Fall 2012 | Home People Lab Write-Up 1 Lab Write-Up 2 Lab Write-Up 3 Course Logistics For Instructors Photos Wiki Editing Help | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
OUR TEAMLAB 1 WRITE-UPPCR Machines allow us to make copies of different DNA samples by heating the DNA to the point of denaturing. Once the two strands of DNA are separated, an enzyme builds complimentary strands using the original strands as a template. The PCR Machine is able to be programmed to running multiple cycles of this heating phase. Each cycle results in doubling the previous amount, so by the end of the 30th cycle, you have just over 1,000,000,000 (one billion) copies of the original DNA. This allows the DNA to be analyzed and tested for any defects.
The Open PCR Machine we received was number 13. When we unplugged the PCB Board of the LCD from the Open PCR Circuit Board, the machine's LCD screen turned off. When we unplugged the white wire that connects the Open PCR Circuit Board to the 16 Tube PCR Block, the machine could not register or measure the temperature. The LCD screen displayed seemingly random numbers including -40 degrees Celsius (which is not possible because the Open PCR Machine was not changing the temperature at that time).
On October 18, 2012, our group first tested the Open PCR Machine number 13. At first, the machine seemed overwhelming in its design. However, after following the instructions and advice from peers and professors, we were able to determine how to properly setup, program, and run a simple test.
ProtocolsPolymerase Chain Reaction Polymerase Chain Reaction is a technology that amplifies a single piece of DNA. This technology works very similarly to the natural DNA replication cycle. One PCR cycle consists of three basic steps, denaturation, annealing and extension. In the denaturation step, heat (usually about 95 degrees Celsius) is used to separate the DNA into two strands. Then in the annealing step, the temperature is decreased to 50 degrees Celsius and the DNA primer, specific to the target sequence for that organism, anneal to the separated strand of DNA. The primers mark the beginning and the end of the targeted DNA sequence. Finally, the extension step required the temperature to be raised to 72 degrees Celsius so that the DNA polymerase is activated. The DNA polymerase begins synthesis at the DNA primer. This results in two double stranded target DNA sequences. The PCR cycle is repeated many times to amplify the targeted strand. There are typically many cycles that need to take place in the PCR in order to amplify a patient's DNA.
After the DNA has been through the thermal cycler, mix each new DNA sample with the the PCR master mix (Taq DNA polymerase, dNTP's, MgCl2, forward primer, and reverse primer) into 8 different Eppendorf tubes using separate pipettes to reduce contamination (see Table 1).
Flourimeter Measurements
After assembling the fluorimeter, you can now determine if you've amplified the targeted DNA in your PCR experiment. Using the Fluorimeter, you can calculate the relative amount of DNA through fluorescence, which is generated by excitation and emission wavelengths. In order to detect fluorescence when dsDNA is present, you'll be using SYBR Green I because it's more safer compared to other dyes. With that being said, gloves must be worn when handling any liquid containing SYBR Green I. The fluorimeter itself is a very simple machine because it uses optical caustic, a special type of optics that completely removes the need for lasers, mirrors, or lenses. Also the flourimeter is battery-powered, lightweight and portable; this allows every student to have one of these at their lab table. Following the steps below, you can easily learn how to dye your amplified DNA.
Research and DevelopmentSpecific Cancer Marker Detection - The Underlying Technology The reason that the cancer-associated sequence of r17879961 will produce a DNA signal while the non-cancer DNA sequence of the same SNP (single nucleotide polymorphism) will not produce a DNA signal lies in the arrangement of nucleotides at the molecular level. The lack of a DNA signal is due to the inability of the reverse primer to bind to the forward strand during the annealing phase of PCR. To detect the cancer-associated sequence of r17879961, the reverse primer AAC TCT TAC ACT CGA TAC AT is used. This is because the cancer-associated mutation is represented by a single nucleotide in a particular triplet: instead of the normal ATT, the middle T mutates into a C, thus rendering a triplet of ACT (which you can see in the reverse primer shown above). At the protein level, this mutation of 1 nucleotide changes the coded protein from isoleucine to threonine. As a result, the primer will not attach to the normal r17879961 DNA sequence as it will not have the corresponding base pairs (TGA) in the particular section of DNA that the mutated sequence would have. (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 OpenPCR tutorial might be useful. Be sure to credit the source if you borrow images.)
Results
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