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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: use each only once. Never re-use disposable pipette tips or samples will be cross contaminated
- Cup for discarded tips
- Micropipettor
- Open PCR machine: shared by two groups
PCR Reaction Sample List
| Tube Label |
PCR Reaction Sample |
Patient ID
|
| G#11PC + |
Positive control |
none
|
| G#11NC - |
Negative control |
none
|
| G#11 1-1 |
Patient 1, replicate 1 |
37595
|
| G#11 1-2 |
Patient 1, replicate 2 |
37595
|
| G#11 1-3 |
Patient 1, replicate 3 |
37595
|
| G#11 2-1 |
Patient 2, replicate 1 |
32179
|
| G#11 2-2 |
Patient 2, replicate 2 |
32179
|
| G#11 2-3 |
Patient 2, replicate 3 |
32179
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DNA Sample Set-up Procedure
- Step 1 : Add DNA samples to separate PCR reaction tubes using different pipette tips for each sample
- Step 2 : Add 50μL of the DNA primer mix to each PCR tube containing a DNA sample
- Step 3 : Add 50μL of the PCR reaction mix to each PCR tube containing a DNA sample
- Step 4 : Put each of the samples into 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, and Extend at 72°C for 30
seconds
- FINAL STEP: 72°C for 2 minutes
- FINAL HOLD: 4°
Research and Development
PCR - The Underlying Technology
Functions of PCR Components
| Template DNA |
This is the DNA from which the the PCR will replicate a particular sequence of interest
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| Primers |
These nucleic acids attach themselves to opposite ends of the sequence of interest once the DNA double helix has been unwound. They provide a marker to indicate where replication should begin.
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| Taq Polymerase |
This enzyme is responsible for attaching free nucleotides in a complementary order to the sequence of interest in the single DNA strand.
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Thermal Cycling of PCR Components
| Initial Step: 95°C for 3 minutes |
Solution is heated to begin denaturing of DNA molecules.
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| Denature at 95°C for 30 seconds |
Double helix of DNA molecules is unwound, leaving single strands of DNA in the solution.
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| Anneal at 57°C for 30 seconds |
Primers attach themselves to the ends of the sequence of interest in each DNA strand.
|
| Extend at 72°C for 30 seconds |
Taq Polymerase attaches at the site of the DNA where the primer is.
|
| Final Step: 72°C for 3 minutes |
Taq Polymerase molecules begin replication by attaching free nucleotides in a complementary order to the single-strand DNA molecules.
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| Final hold: 4°C |
Solution is cooled to allow the single-strand DNA molecules to reform into their original double helix shape, leaving many fully-formed copies of the desired sequence.
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BME 100 Spring 2015
