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Team:Hawaii/Ligation of pRL1383a Parts
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Contents |
Ligation of Parts
- The BioBrick parts of pRL1383a are to be ligated in a series of experiments.
Methods
Restriction Digest
- Each part is digested with both enzymes. For all cases NEBuffer 2 is used.
- Reaction Conditions:
- 5ul Buffer
- 1ul Enzyme 1 + 1ul Enzyme 2
- 0.5 ul BSA
- Xul water
- Yul insert (1ug if possible)
- Zul vector (less than 1ug)
- Refer to http://openwetware.org/wiki/DNA_ligation for the reasoning behind this.
- Running Conditions: 2 hours at 37°C.
- Check the progress of the reaction by running a 0.8% gel.
- Ligation
| Name |
|---|
| rep(80ng)+B0030(25ng) |
| oriV(20ng)+pSB1A2(50ng) |
| aadA (BB) |
- Transformation
- Verification with Colony PCR
Results
8/11
| Name | size | enzyme | quantity | date of digest |
|---|---|---|---|---|
| rep | 3.3kb | XbaI & PstI | 5ng/ul | 8/11/08 |
| oriV | 415bp | XbaI & PstI | 5ng/ul | 8/11/08 |
| aadA (pRL1383a) | 806bp | XbaI & PstI | 5ng/ul | 8/11/08 |
| aadA (BB) | 806bp | XbaI & PstI | 5ng/ul | 8/11/08 |
| oriT | ~125bp | XbaI & PstI | n/a | 8/27/08 |
| P1 lytic Region | 1.3kb | EcoRI & SpeI | 5ng/ul | 8/11/08 |
| [http://partsregistry.org/Part:BBa_B0030 BBa_B0030] | 2094bp | SpeI & PstI | 4ng/ul | 8/11/08 |
| [http://partsregistry.org/Part:BBa_B0015 BBa_B0015] | 3318bp | EcoRI & XbaI | 5.7ng/ul | 8/11/08 |
| [http://partsregistry.org/Part:pSB1A2 pSB1A2] | 2094bp | SpeI & PstI | 5.5ng/ul | 8/11/08 |
| [http://partsregistry.org/Part:BBa_I14032 BBa_I14032] | 4462bp | n/a | n/a |
Discussion
- What was learned and how to do future experiments differently.
[http://manoa.hawaii.edu/ 
][http://manoa.hawaii.edu/ovcrge/ 
][http://www.ctahr.hawaii.edu 
]
