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| - | Descriptive Title of What You're Doing
| + | PCR Amplification of LuxPQ |
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| | <div class="desc"> | | <div class="desc"> |
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| - | WIKI CODING HERE
| + | <b>Purpose:</b> |
| | + | <br> |
| | + | To amplify LuxPQ on TOPO II Blunt on a PCR and verify that the sample really does consist of LuxPQ. |
| | + | |
| | + | <b>Materials and methods:</b> |
| | + | <br> |
| | + | *DNA template: LuxPQ on TOPO II blunt |
| | + | *Forward primer: LuxPQ forward (T_m = 60 degrees Celsius) |
| | + | *Reverse primer: LuxPQ reverse (T_m = 60 degrees Celsius) |
| | + | *Expected PCR product size: 3855 bp |
| | + | *DNA template concentration: |
| | + | **Colony 1: 254.7 ng/uL |
| | + | **Colony 9: 168.7 ng/uL |
| | + | <br> |
| | + | 2 amplification tubes were made for each colony, in addition to a negative control tube (for a total of 5 tubes) |
| | + | <br> |
| | + | Master Mix contents (for 5 tubes): |
| | + | <br> |
| | + | |
| | + | *10X PCR Buffer minus Mg 2+ (25 uL) |
| | + | *10mM dNTPs mixture (5 uL) |
| | + | *50mM MgCl2 (7.5 uL) |
| | + | *Forward primer [10 uM] (7.5 uL) |
| | + | *Reverse primer [10 uM] (7.5 uL) |
| | + | *Taq DNA polymerase [5 units/uL] (2.0 uL) |
| | + | *Autoclaved distilled HOH (190.5 uL) |
| | + | <br> |
| | + | <b>TOTAL:</b> 245 uL of Master Mix, for 5 tubes of 49 uL each. |
| | + | <br> |
| | + | |
| | + | Each PCR tube requires between 100-200 ng of template DNA. Accordingly, the Colony 1 sample was diluted to 200 ng/uL. 1 uL of DNA template was added to the following tubes: |
| | + | *Tube 1: Colony 1 |
| | + | *Tube 2: Colony 1 |
| | + | *Tube 3: Colony 9 |
| | + | *Tube 4: Colony 9 |
| | + | *Tube 5: dd HOH (negative control). |
| | + | <br> |
| | + | PCR steps: |
| | + | *Denaturation: 94 degrees C, 3 minutes |
| | + | *Amplification: 36 cycles of: |
| | + | **Denaturation (94 degrees C, 45 seconds); |
| | + | **Annealing (55 degrees C, 45 seconds); |
| | + | **Extension (72 degrees C, 4 minutes)} |
| | + | *Final extension: 72 degrees C, 10 minutes |
| | + | *Hold temperature: 4 degrees C |
| | + | |
| | + | <b>Results:</b> |
| | + | None to offer here – the PCR tubes had mysteriously vanished from the PCR machine when I returned to collect them after the PCR had finished. |
| | + | |
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CAROL
Amplification of luxCDABE using Taq polymerase
The purpose of this experiment was to amplify luxCDABE with Taq polymerase. Gene specific luxCDABE forward and reverse primers were used. Conditions used were: Denature (94 Degrees Celsius, 3 minutes), 30 cycles (Denature (94 Degrees Celsius, 45 Seconds), Anneal (52 Degrees Celsius, 45 Seconds), Extend (72 Degrees Celsius, 6 minutes)), Final Extension (72 Degrees Celsius, 10 minutes), and hold at 4 Degrees Celsius overnight.
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CHINMOYEE
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EMILY
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FAHD
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IMAN
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JAMIE
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JEREMY
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KATIE
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KEVIN
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MANDY
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PATRICK
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PRIMA
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STEFAN
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VICKI
PCR Amplification of LuxPQ
Purpose:
To amplify LuxPQ on TOPO II Blunt on a PCR and verify that the sample really does consist of LuxPQ.
Materials and methods:
- DNA template: LuxPQ on TOPO II blunt
- Forward primer: LuxPQ forward (T_m = 60 degrees Celsius)
- Reverse primer: LuxPQ reverse (T_m = 60 degrees Celsius)
- Expected PCR product size: 3855 bp
- DNA template concentration:
- Colony 1: 254.7 ng/uL
- Colony 9: 168.7 ng/uL
2 amplification tubes were made for each colony, in addition to a negative control tube (for a total of 5 tubes)
Master Mix contents (for 5 tubes):
- 10X PCR Buffer minus Mg 2+ (25 uL)
- 10mM dNTPs mixture (5 uL)
- 50mM MgCl2 (7.5 uL)
- Forward primer [10 uM] (7.5 uL)
- Reverse primer [10 uM] (7.5 uL)
- Taq DNA polymerase [5 units/uL] (2.0 uL)
- Autoclaved distilled HOH (190.5 uL)
TOTAL: 245 uL of Master Mix, for 5 tubes of 49 uL each.
Each PCR tube requires between 100-200 ng of template DNA. Accordingly, the Colony 1 sample was diluted to 200 ng/uL. 1 uL of DNA template was added to the following tubes:
- Tube 1: Colony 1
- Tube 2: Colony 1
- Tube 3: Colony 9
- Tube 4: Colony 9
- Tube 5: dd HOH (negative control).
PCR steps:
- Denaturation: 94 degrees C, 3 minutes
- Amplification: 36 cycles of:
- Denaturation (94 degrees C, 45 seconds);
- Annealing (55 degrees C, 45 seconds);
- Extension (72 degrees C, 4 minutes)}
- Final extension: 72 degrees C, 10 minutes
- Hold temperature: 4 degrees C
Results:
None to offer here – the PCR tubes had mysteriously vanished from the PCR machine when I returned to collect them after the PCR had finished.
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