BME103:T130 Group 14 l2: Difference between revisions
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! scope="col" | Reagent | |||
! scope="col" | Volume | |||
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| Template DNA ( 20 nanograms) | |||
| 0.2 microliters | |||
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| 10 micrometers forward primer | |||
| 1.0 microliters | |||
|- | |||
| 10 micrometers reverse primer | |||
|1.0 microliters | |||
|- | |||
| GoTaq Master Mix | |||
| 50.0 microliters | |||
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| Distilled Water | |||
| 47.8 microliters | |||
|- | |||
|Total Volume | |||
|100.0 microliters | |||
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'''PCR Protocol''' | '''PCR Protocol''' | ||
<br><br> | |||
'''Setting up your Tubes''' | |||
#Collect 8 samples of DNA and 1 DNA(calf thymus standard at 2 micrograms/mL) sample and water from the scintillation vial to analyze. | |||
#With a permanent marker, number the transfer pipettes at the bulb so that you limit cross-contamination and only use '''ONE''' per solution, and number your Eppendrof tubes at the top. At the end, you should have 10 Eppendorf tubes and 10 pipettes clearly labeled. | |||
#Transfer each sample separately ( using one pipette per sample) into an Eppendorf tube containing 400 mL of buffer. Label this tube with the number of your sample. Make sure to transfer all of the sample into the Eppendorf tube. Use the same pipette to place ONLY this sample's drop onto the fluorescent measuring device. | |||
#Take the specially labeled Eppendorf tube containing the SYBR GREEN I and using it's assigned pipette, place two drops on the first two centered drops. | |||
#Now take the diluted sample and place two drops on top of the SYBR GREEN I solution drops. | |||
# Align the light going through the drop. | |||
# Take pictures with the smartphone, as many as desired. | |||
# If you are not satisfied with that sample, you may rerun that sample again or move on to the next sample. | |||
# Be sure to only run 5 samples per slide. | |||
# Before completing the lab, run the water from the scintillation vial as a BLANK using the same procedure listed above. | |||
<br><br> | |||
'''PCR Machine''' | |||
#Acquire the DNA samples that have been submitted for testing <br> | |||
#Run the DNA samples through the thermal cycler program (see '''Stage 1''') <br> | |||
##'''Stage 1 (Initiation):''' 1 cycle at 95°C for 3 minutes, to separate the DNA strand. <br> | |||
##'''Stage 2 (Denaturation):''' 35 cycles: first at 95°C for 30 seconds, and then gradually decrease the temperature to approximately 57°C for 30 seconds, and then raise the temperature to approximately 72°C for 30 seconds, so the DNA polymerase can be activated. This is also an example of heat shock, and is effective to initiate the addition of complementary nucleotides onto the DNA strand, which the DNA polymerase does. <br> | |||
##'''Stage 3 (Elongation) :''' At this step, hold the DNA at 72°C for 3 minutes.The temperature is held here so that the DNA polymerase can copy the strand. Also, this is where 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 began with. <br> | |||
##'''Stage 4:''' Final hold until the DNA stabilizes at 4°C. At the end of this cycle you will have 8 fragments of the DNA<br> | |||
#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 | |||
'''DNA Measurement Protocol''' | '''DNA Measurement Protocol''' |
Revision as of 14:04, 29 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 2 WRITE-UPThermal Cycler EngineeringOur re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski.
Key Features -Improved fiberglass insulation Instructions
ProtocolsMaterials
PCR Protocol
DNA Measurement Protocol Research and DevelopmentBackground on Disease Markers
Primer Design
Illustration
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