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Team:Newcastle/19 August 2010
From 2010.igem.org
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Contents |
Gel extraction and ligation of filamentous cell part into pGFPrrnB and pSB1C3
Aims
The aim of the experiment is to further purify the filamentous cell part (yneA), pGFPrrnB and pSB1C3 by doing gel extraction. The purified fragments are then ligated together.
Materials and Protocol
Please refer to gel electrophoresis, gel extraction, nanodrop spectrophotometer and ligation.
Ligation mix:
| Reagents | 1:3(μl) | 1:5(μl) | Vector(μl) |
|---|---|---|---|
| Vector | 3 | 2 | 1.5 |
| Insert | 3 | 6 | 7.5 |
| 10X BUFFER | 1 | 1 | 1.1 |
| T4 Ligase | 1 | 1 | 1 |
| H2O | 2 | 0 | 0 |
| Total Volume | 10.0 | 10.0 | 10.0 |
Table 1: Ligation mix for ligation of yneA into pGFPrrnB and pSB1C3
Results
| yneA 1 | yneA 2 | yneA 3 | pSB1C3 | pGFPrrnB | |
|---|---|---|---|---|---|
| Concentration of DNA ng/µl | 3.4 | 4.7 | 5.4 | 2.2 | 18.5 |
Table 2: Nanodrop spectrophotometer results. Table represents the amount of plasmid present in µl/ml quantity.
Discussion
The DNA concentration obtained for yneA ranged from 3.4 µl/ml to 5.4 µl/ml, 2.2 µl/ml for pSB1C3 and 18.5 µl/ml for pGFPrrnB . Therefore the concentration for yneA and pSB1C3 is low.
Conclusion
We will proceed with digestion, but another set of ligation will be set up to repeat the process again.
Results and Conclusion
Ligation will be done overnight because concentration of the filamentous cell part, pGFPrrnB and pSB1C3 are not very good. Please refer to 20.08.10 for results and discussion. Overnight culture of yneA, pSB1C3 and pGFPrrnB were set up for miniprep extraction tomorrow, 20th August, 2010.
   
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