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Team:Hawaii/PCR Amplification of pRL1383a
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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 
]
