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Team:Newcastle/19 August 2010
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Please refer to [[Team:Newcastle/PCR| PCR]] for Phusion PCR protocol. | Please refer to [[Team:Newcastle/PCR| PCR]] for Phusion PCR protocol. | ||
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| + | =''rocF'' BioBrick= | ||
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| + | ==Gel extraction of linearised pSB1C3== | ||
| + | ==PCR of pSB1C3, pspac oid and double terminator with new primers== | ||
{{Team:Newcastle/footer}} | {{Team:Newcastle/footer}} | ||
Revision as of 20:37, 19 August 2010
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Contents |
Gel extraction of yneA, pGFPrrnB and pSB1C3
Aims
To extract the correct size bands for yneA, pGFPrrnB and PSB1AT3 from the gel after running gel electrophoresis. Concentration of the DNA and vectors are also checked with nanodrop.
Materials and Protocol
Please refer to gel electrophoresis, gel extraction, nanodrop spectrophotometer.
Results
| yneA 1 | yneA 2 | yneA 3 | pSB1C3 | pGFPrrnB | |
|---|---|---|---|---|---|
| Concentration of DNA ng/µl | 3.4 | 4.7 | 5.4 | 2.2 | 18.5 |
Discussion
The bands we extracted from the gel was good for pGFPrrnB, but not so strong for yneA and PSB1C3.
The results we got from the nanodrop were not as good as expected for yneA and PSB1C3.
Conclusion
We will proceed with digestion, but another set of ligation will be set up to repeat the process again.
Ligation for pGFPrrnB with yneA and pSB1C3 with yneA
Aims
To ligate yneA into both vectors pGFPrrnB and pSB1C3.
Materials and Protocol
Please refer to ligation.
Ligation mix for pSB1C3 with yneA
The concentration of pSB1C3 and yneA that we got from the gel extraction earlier today was very low, so we used a different mixture set up for 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 |
Results and Conclusion
Please refer to 20.08.10.
Digestion of yneA, pGFPrrnB and pSB1C3
Aim
To repeat digestion from 18.08.10.
Materials and Protocol
Please refer to restriction digest.
Setting up Overnight Cultures for miniprep
Aims
To prepare cultures for miniprep of yneA, pGFPrrnB and pSB1C3 tomorrow.
Materials and Protocol
Please refer to growing an overnight culture.
Go back to our main Lab book page
Subtilin Immunity
Aims
We have modified the promoters for Plasmid Vector (part 1), Promoter & RBS (part 2) and Double terminator (part 4), thus we aim to do Phusion PCR today.
Materials and protocol
Please refer to PCR for Phusion PCR protocol.
rocF BioBrick
Gel extraction of linearised pSB1C3
PCR of pSB1C3, pspac oid and double terminator with new primers
   
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