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Team:Newcastle/19 August 2010
From 2010.igem.org
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| - | These primers were ordered as a solution to the problem we encountered when trying to carry out the Gibson protocol for the Subtilin Immunity (and RocF) BioBrick. These primers will be used with previously rehydrated primers to try and obtain all four parts that we need to make the Subtilin Immunity BioBrick using the Gibson Protocol. So far the ''spaIFEG'' coding sequence (part 3) has already been successfully obtained. | + | These primers were ordered as a solution to the problem we encountered when trying to carry out the Gibson protocol for the Subtilin Immunity (and RocF) BioBrick. These primers will be used with previously rehydrated primers to try and obtain all four parts that we need to make the Subtilin Immunity BioBrick using the Gibson Protocol. So far the ''spaIFEG'' coding sequence (part 3) has already been successfully obtained. We still need to obtain the plasmid vector (part 1), promoter & RBS (part 2) and double terminator (part 4). So in order to do this the primers received today will be rehydrated and Phusion PCR will be carried out for each of the parts in order to amplify these parts. |
| + | ==Materials and protocol== | ||
| - | + | Please refer to [[Team:Newcastle/PCR| PCR]] for Phusion PCR protocol. | |
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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. | ||
Revision as of 08:18, 20 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.
Subtilin Immunity
Aims
Today we received four new primers that we ordered:
- Prom_For
- pSB1C3_for
- pSB1C3_rev
- Term_rev.
These primers were ordered as a solution to the problem we encountered when trying to carry out the Gibson protocol for the Subtilin Immunity (and RocF) BioBrick. These primers will be used with previously rehydrated primers to try and obtain all four parts that we need to make the Subtilin Immunity BioBrick using the Gibson Protocol. So far the spaIFEG coding sequence (part 3) has already been successfully obtained. We still need to obtain the plasmid vector (part 1), promoter & RBS (part 2) and double terminator (part 4). So in order to do this the primers received today will be rehydrated and Phusion PCR will be carried out for each of the parts in order to amplify these parts.
Materials and protocol
Please refer to PCR for Phusion PCR 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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