BME103:T130 Group 13: Difference between revisions
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<br>'''Cycle 2:''' the same three steps occurring in cycle happen in cycle 2. The temperature is raised again to separate the DNA strands, the temperature is lowered so that the primers may attach, and the temperature is raised again slightly to stimulate DNA polymerase to copy the strand. | <br>'''Cycle 2:''' the same three steps occurring in cycle happen in cycle 2. The temperature is raised again to separate the DNA strands, the temperature is lowered so that the primers may attach, and the temperature is raised again slightly to stimulate DNA polymerase to copy the strand. | ||
<br>'''Cycle 3:''' the two desired fragments begin to appear—two strands that begin with primer one and end with primer two—and these are the DNA copies of the segment of DNA you’ve targeted. These products will increase (become the majority) as the cycle continues. | <br>'''Cycle 3:''' the two desired fragments begin to appear—two strands that begin with primer one and end with primer two—and these are the DNA copies of the segment of DNA you’ve targeted. These products will increase (become the majority) as the cycle continues. | ||
<br>'''Cycle 4:''' at the end of this cycle, you‘ll have 8 fragments that contain only your target sequence. | <br>'''Cycle 4:''' at the end of this cycle, you‘ll have 8 fragments that contain only your target sequence ('''see Table 2'''). | ||
<br>'''Cycle 5:''' at the end of this cycle, you‘ll have 22 fragments that your target sequence and only ten longer length copies. | <br>'''Cycle 5:''' at the end of this cycle, you‘ll have 22 fragments that your target sequence and only ten longer length copies. | ||
<br>'''After 30 cycles''' there are over a billion fragments that contain only your target sequence and only 60 copies of the longer length molecules. You now have a solution of nearly pure target sequence. | <br>'''After 30 cycles''' there are over a billion fragments that contain only your target sequence and only 60 copies of the longer length molecules. You now have a solution of nearly pure target sequence. | ||
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). | 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'''). | ||
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<br> | '''Table 2''' | ||
<br> | |||
{| border="1" cellpadding="5" cellspacing="0" align="center" | |||
! scope="col" | '''DNA Sample Descriptions (8 Samples)''' | |||
|- | |||
| Positive Control: Cancer DNA Template | |||
| Patient 1 Replicate 1: 65685 | |||
| Patient 1 Replicate 2: 65685 | |||
| Patient 1 Replicate 2: 65685 | |||
|- | |||
| Negative Control: No DNA Template | |||
| Patient 2 Replicate 1: 58278 | |||
| Patient 2 Replicate 2: 58278 | |||
| Patient 2 Replicate 3: 58278 | |||
|- | |||
|} | |||
'''Flourimeter Measurements'''<br> | '''Flourimeter Measurements'''<br> | ||
<br>'''Fluorimeter Assembly Procedure''' | <br>'''Fluorimeter Assembly Procedure''' | ||
<br>1) Turn on the excitation light using the switch for the blue LED. | <br>1) Turn on the excitation light using the switch for the blue LED. | ||
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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. | 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>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>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>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>4)Take Eppendorf tube labelled SYBR Green I and using the specially labeled pipette, place 2 drops on the first two centered drops. | ||
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<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>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>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 21:41, 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).
Table 2
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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