OUR TEAM
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LAB 4 WRITE-UP
Protocol
Materials
- Lab Coat
- Disposable Gloves
- PCR Reaction Mix, 8 tubes, 50 uL each: Mix contatins Taq DNA polymerase, MgCL2, and dNTP's
- DNA/primer mix, 8 tubes, 50 uL 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 machines: shared by two groups
PCR Reaction Sample List
Tube Label |
PCR Reaction Sample |
Patient ID
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G8 P |
Positive control |
none
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G8 N |
Negative control |
none
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G8 1-1 |
Patient 1, replicate 1 |
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G8 1-2 |
Patient 1, replicate 2 |
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G8 1-3 |
Patient 1, replicate 3 |
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G8 2-1 |
Patient 2, replicate 1 |
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G8 2-2 |
Patient 2, replicate 2 |
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G8 2-3 |
Patient 2, replicate 3 |
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DNA Sample Set-up Procedure
- Acquire the DNA samples from the patients as well as PCR reaction mix
- Label all of the PCR tubes for all of the samples that will be tested
- Place a new pipette tip on the micropipette
- Take 50 microliters of the PCR reaction mix
- Transfer PCR reaction mix to the corresponding labeled empty PCR tube
- Discard the used pipette tip
- Put a new pipette tip on the micropipette
- Take 50 micro liters of patient DNA sample/primer corresponding to the label on the tube
- Transfer the patient DNA into the tube containing the PCR reaction mix
- Repeat steps 3 through 9 for all samples. Be sure to place the DNA in the correct labeled tubes
- Place the tubes in the thermal cycler.
OpenPCR program
Heated Lid: 100°C
Initial Step: 95°C for 2 minutes
Number of Cycles: 35
Denature at 95°C for 30 seconds
Anneal at 57°C for 30 seconds
Extend at 72°C for 30 seconds
Final Step: 72°C for 2 minutes
Final Hold: 4°C
Research and Development
PCR - The Underlying Technology
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