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| - | Descriptive Title of What You're Doing
| + | Planning PCR Activity |
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| - | WIKI CODING HERE
| + | Started to make an outline of what I will be required to do for PCR machine: |
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| | + | * Need some way to add items to the machine to make it more interactive then just telling the machine what is required |
| | + | * Looked through the lsl wiki and found llAllowInventoryDrop, which is a function that seems to allow for addition of objects to another as long as it is set to true |
| | + | * If anything can be added then I need to have a way to get rid of everything that is added |
| | + | * I will need some way to incorporate other information besides materials, such as temperatures and times for each cycle |
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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
Descriptive Title of What You're Doing
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EMILY
Descriptive Title of What You're Doing
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FAHD
Descriptive Title of What You're Doing
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IMAN
Descriptive Title of What You're Doing
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JAMIE
Descriptive Title of What You're Doing
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JEREMY
Descriptive Title of What You're Doing
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KATIE
Planning PCR Activity
Started to make an outline of what I will be required to do for PCR machine:
- Need some way to add items to the machine to make it more interactive then just telling the machine what is required
- Looked through the lsl wiki and found llAllowInventoryDrop, which is a function that seems to allow for addition of objects to another as long as it is set to true
- If anything can be added then I need to have a way to get rid of everything that is added
- I will need some way to incorporate other information besides materials, such as temperatures and times for each cycle
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KEVIN
Descriptive of What You're Doing
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MANDY
Descriptive Title of What You're Doing
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PATRICK
Descriptive Title of What You're Doing
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PRIMA
Descriptive Title of What You're Doing
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STEFAN
Descriptive Title of What You're Doing
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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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