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OUR TEAM
LAB 4 WRITE-UP
Protocol
Materials
- Lab coat and disposable gloves
- PCR reaction mix, 8 tubes, 50 μL each: Mix contains Taq DNA polymerase, MgCl2, and dNTP’s
- DNA/primer mix, 8 tubes, 50 μL each: Each mix contains a different template DNA. All tubes have the same forward primer and reverse primer
- A strip of empty PCR tubes
- Disposable pipette tips: only use each only once. Never re-use disposable pipette tips or samples will be cross-contaminated
- Cup for discarded tips
- Micropipettor
- OpenPCR machine: shared by two groups
PCR Reaction Sample List
| Tube Label |
PCR Reaction Sample |
Patient ID
|
| G# + |
Positive control |
none
|
| G# - |
Negative control |
none
|
| G# 1-1 |
Patient 1, replicate 1 |
83526
|
| G# 1-2 |
Patient 1, replicate 2 |
83526
|
| G# 1-3 |
Patient 1, replicate 3 |
83526
|
| G# 2-1 |
Patient 2, replicate 1 |
31092
|
| G# 2-2 |
Patient 2, replicate 2 |
31092
|
| G# 2-3 |
Patient 2, replicate 3 |
31092
|
DNA Sample Set-up Procedure
- Start by acquiring the necessary equipment for the laboratory experiment, including safety goggles, disposable gloves and lab coat.
- Two buckets will be filled to the brim with ice.
- Place the PCR reaction mix (8 tube setup, 50 microliters each, mixture will contain Taq polymerase, dNTP's and magnesium chloride) into one bucket of ice.
- Place the DNA and primer mix (8 tube setup, 50 microliters each, different templates of DNA per tube) into the same bucket of ice.
- Place the set of empty PCR tubes into the other bucket of ice.
- Using the micropipette and a disposable tip, extract 50 microliters of the PCR reaction mix and place into an empty PCR tube. Dispose of the tip into the cup after the 50 microliter collection has been deposited into the PCR tube.
- With a new disposable tip, extract 50 microliters of the DNA and primer mix, and place into the same tube that contains the PCR reaction mix. Dispose of the tip into the cup after the 50 microliter collection has been deposited into the PCR tube.
- This process will repeat steps 6 and 7 for each of the remaining seven PCR tubes.
OpenPCR program
HEATED LID: 100°C
- With the PCR tubes filled, place them into the OpenPCR machine when the device has reached the above temperature of 100 degrees Celsius.
INITIAL STEP: 95°C for 2 minutes
- Reduce the temperature to 95 degrees Celsius, and allow the PCR tubes to heat for two minutes.
DENATURE: 95°C for 30 seconds
- At 95 degrees Celsius, have the PCR tubes denatured for 30 seconds.
ANNEAL: 57°C for 30 seconds
- Reduce the temperature to 57 degrees Celsius in order to allow the contents of the PCR tubes to anneal.
EXTEND: 72°C for 30 seconds
- Increase the temperature to 72 degrees Celsius to initiate extending.
NUMBER OF CYCLES: 35
- The DENATURE, ANNEAL AND EXTEND phases will occur for thirty-five cycles.
FINAL STEP: 72°C for 2 minutes
- Allow DNA replication to occur for two minutes.
FINAL HOLD: 4°C
- Reduce the temperature to 4 degrees Celsius, and the PCR tubes can be removed.
Research and Development
PCR - The Underlying Technology
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BME 100 Spring 2015
