BME103:T130 Group 14: Difference between revisions
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#After the DNA has been through the numerous cycles, you will have over thousands of fragments of the same DNA sequences. After the DNA has been through the thermal cycle, mix each DNA sample with the PCR reaction mix (Taq DNA polymerase, MgCl2, dNTP’s, and a forward and reverse primer), using a separate pipette each time to reduce cross-contamination into 8 separate tubes (see '''Table 2'''). | #After the DNA has been through the numerous cycles, you will have over thousands of fragments of the same DNA sequences. After the DNA has been through the thermal cycle, mix each DNA sample with the PCR reaction mix (Taq DNA polymerase, MgCl2, dNTP’s, and a forward and reverse primer), using a separate pipette each time to reduce cross-contamination into 8 separate tubes (see '''Table 2'''). | ||
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==Results== | ==Results== | ||
<!--- Place two small Image J data images here. One showing the result of Water and the other showing the result of Calf Thymus DNA ---> | |||
<!--- Enter the values from your group's Data Analyzer table below. E6, F6, etc. are the excel cells from which you should copy your data. ---> | |||
{| {{table}} | |||
|- style="background:#f0f0f0;" | |||
| '''Sample''' || '''Integrated Density''' || '''DNA μg/mL''' || '''Conclusion''' | |||
|- | |||
| PCR: Negative Control || E6 || F6 || G6 | |||
|- | |||
| PCR: Positive Control || E7 || F7 || G7 | |||
|- | |||
| PCR: Patient 1 ID #####, rep 1 || E8 || F8 || G8 | |||
|- | |||
| PCR: Patient 1 ID #####, rep 2 || E9 || F9 || G9 | |||
|- | |||
| PCR: Patient 1 ID #####, rep 3 || E10 || F10 || G10 | |||
|- | |||
| PCR: Patient 2 ID #####, rep 1 || E11 || F11 || G11 | |||
|- | |||
| PCR: Patient 2 ID #####, rep 2 || E12 || F12 || G12 | |||
|- | |||
| PCR: Patient 2 ID #####, rep 3 || E13 || F13 || G13 | |||
|} | |||
KEY | |||
* '''Sample''' = <!--- explain what "sample" means ---> | |||
* '''Integrated Density''' = <!--- explain what "integrated density" means and how you did background subtraction to get this value ---> | |||
* '''DNA μg/mL''' = <!--- explain how you calculated this ---> | |||
* '''Conclusion''' = <!--- explain what "Positive" and "No signal" means, relative to the control samples ---> | |||
Revision as of 14:55, 9 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-UPInitial Machine TestingThe Original Design The Open PCR machine is fairly easy to use. Simply plug it in to the USB port on your computer and turn it on. From your computer, you can adjust the temperature, add or remove steps to the process, and adjust the number of cycles.
When we unplugged the LCD from the Circuit Board, the LCD turned off. When we unplugged the white wire that connects the circuit board to heat block, the LCD was unable to accurately read the temperature.
The very first time that our group used the Open PCR machine was October 28, 2012. The process went fairly smoothly, which was unexpected. All we did was put the samples into the machine, set the peak temperature and number of cycles, then let it run for an hour and forty-nine minutes.
ProtocolsPolymerase Chain Reaction The polymerase chain reaction (PCR) is a biochemical machine used in biological chemistry to produce numerous copies of a particular piece of DNA, generating multiple duplicates of DNA sequences. The PCR machine works similar the cycle of DNA replication at the cellular level. The machine consists of four individual steps, initiation, denaturation, annealing, and extension. The initiation step is solely to prepare the DNA samples to be put through the thermal cycler program. During the denaturation step, the DNA strand is split into two separate strands. After, the annealing step is where the DNA primer attaches to the targeted DNA sequence. The primer only attached to a specific site on the strand, not necessarily the entire strand. The purpose of the primer is to mark the beginning and the end of the targeted DNA sequence. Lastly, in the extension step, the DNA polymerase is first activated, which begins to synthesize the DNA primer. This results in two double stranded target DNA sequences. The PCR machine cycles numerous times to amplify the specific sequence. In order to complete the reaction several components are required such as:
Table 1
Table 2
Type of smartphone used: Iphone 4s In this lab, we used SYBR Green I, which is used to detect a particular DNA strand. In this lab, our fluorimeter was created using optical caustic because it eliminated the need for lasers, mirrors, and lenses. A fluorimeter is a device used to measure parameters of fluorescence: it's intensity and and wavelength distribution. The quality is related to the amount of florescent material and indirectly proportional to the molecule being detected. The objective of this part is to determine if you have actually amplified the target DNA in your PCR experiment. Through this you will be able to visually observe the fragments and calculate the relative amount of DNA. During the first procedure, you amplified your DNA, so now that you are going dye it with the following procedure:
Research and DevelopmentSpecific Cancer Marker Detection - The Underlying Technology (Add a write-up of the information discussed in Week 3's class) (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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