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Team:Hawaii/PCR Amplification of pRL1383a
From 2008.igem.org
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|+ '''<strong>PCR Running Conditions</strong>''' | |+ '''<strong>PCR Running Conditions</strong>''' | ||
| - | !width=" | + | !width="150"|Duration of Time |
| - | !width=" | + | !width="150"|T° 5 cycles |
| - | !width=" | + | !width="150"|T° 30 cycles |
| - | !width=" | + | !width="150"|Purpose |
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|10 minutes | |10 minutes | ||
| - | |95& | + | |95°C |
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|heat activate taq | |heat activate taq | ||
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|30 seconds | |30 seconds | ||
|95°C | |95°C | ||
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|denaturation | |denaturation | ||
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|5 minutes | |5 minutes | ||
|68°C | |68°C | ||
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|extension (1.5 minutes per kb, go with longest) | |extension (1.5 minutes per kb, go with longest) | ||
|- | |- | ||
|10 minutes | |10 minutes | ||
|68°C | |68°C | ||
| - | | | + | |68°C |
|finishing the extension | |finishing the extension | ||
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|infinity | |infinity | ||
|4°C | |4°C | ||
| - | | | + | |4°C |
|(until you remove product) | |(until you remove product) | ||
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Revision as of 23:47, 3 July 2008
| Projects | Events | Resources | ||
|---|---|---|---|---|
| Sponsors | Experiments | Milestones | Protocols | |
| Notebook (t) | Meetings (t) |
Contents |
PCR Amplification of pRL1383a
- Several regions of pRL1383a will be amplified with BioBrick based primers. These components will be used later in the construction of a pRL1383a BioBrick based vector. These parts include the aadA region of the omega interposon, the origin of vegetative replication (oriV), the mobilization proteins, and the replication proteins.
Methods
Materials
- Taq polymerase: Accusure(hot start, 68°C) and Red Taq
- Nanopure water
- forward/reverse primers for each BioBrick
- pRL1383a as template
| Duration of Time | T° 5 cycles | T° 30 cycles | Purpose |
|---|---|---|---|
| 10 minutes | 95°C | heat activate taq | |
| 30 seconds | 95°C | 95°C | denaturation |
| 30 seconds | 48.1°C, 53.4°C, 57.9°C | 59°C, 60.6°C, 61.9°C | annealing |
| 5 minutes | 68°C | 68°C | extension (1.5 minutes per kb, go with longest) |
| 10 minutes | 68°C | 68°C | finishing the extension |
| infinity | 4°C | 4°C | (until you remove product) |
Results
Discussion
- What was learned and how to do future experiments differently.
Insanity is doing the same thing over and over again and expecting different results. - Albert Einstein
[http://manoa.hawaii.edu/ 
][http://manoa.hawaii.edu/ovcrge/ 
][http://www.ctahr.hawaii.edu 
]
