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Team:Newcastle/19 August 2010
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(→Gel extraction of linearised pSB1C3 for the rocF BioBrick) |
RachelBoyd (Talk | contribs) (→Gel extraction and ligation of yneA into pGFPrrnB and PSB1AT3) |
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{{Team:Newcastle/mainbanner}} | {{Team:Newcastle/mainbanner}} | ||
| - | =Gel extraction and ligation of ''yneA'' into pGFPrrnB and | + | =Gel extraction and ligation of ''yneA'' into pGFPrrnB and pSB1C3= |
==Aims== | ==Aims== | ||
| - | The aim of the experiment is to further purify the ''yneA'', pGFPrrnB and | + | The aim of the experiment is to further purify the ''yneA'', pGFPrrnB and pSB1C3 by doing gel extraction. The purified fragments are then ligated together. |
===Materials and Protocol=== | ===Materials and Protocol=== | ||
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Ligation will be done overnight because concentration of ''yneA'', pGFPrrnB and pSB1C3 are not very good. Please refer to [[Team:Newcastle/20_August_2010|20.08.10]] for results and discussion. Overnight culture of ''yneA'', pSB1C3 and pGFPrrnB were set up for miniprep extraction tomorrow, 20th August, 2010. | Ligation will be done overnight because concentration of ''yneA'', pGFPrrnB and pSB1C3 are not very good. Please refer to [[Team:Newcastle/20_August_2010|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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=Rehydration of new subtilin immunity primers and PCR amplification= | =Rehydration of new subtilin immunity primers and PCR amplification= | ||
Revision as of 18:06, 27 October 2010
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Contents |
Gel extraction and ligation of yneA into pGFPrrnB and pSB1C3
Aims
The aim of the experiment is to further purify the 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 PSB1AT3
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 yneA, 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.
Rehydration of new subtilin immunity primers and PCR amplification
Aims
The aim of the experiment is to rehydrate the new primers, Prom_For, pSB1C3_for, pSB1C3_rev and Term_rev. The primers is then used to amplify the spaIFEG gene cluster, Promoter and RBS (pVeg-SpoVG) and Double terminator .
These primers were ordered as a solution to the problem we encountered before we were about to attempt carry out the Gibson protocol for the Subtilin Immunity (and RocF) BioBrick. The 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 DNA rehydration page for the protocol for the rehydration of the four primers and to the Phusion PCR page for protocol followed for Phusion PCR.
| Tube | Part to be amplified | DNA fragment consisting the part | Forward primer | Reverse Primer | Melting Temperature (Tm in °C) | Size of the fragment (in bp) | Extension time* (in seconds) |
|---|---|---|---|---|---|---|---|
| 1 | Plasmid Vector | pSB1C3 | pSB1C3_for | pSB1C3_rev | 68 | 2046 + | 70 |
| 2 | Promoter and RBS (pVeg-SpoVG) | BioBrick Bba_K143053 | Prom_for | P2P2_rev | 67 | 139 + | 15 |
| 4 | Double terminator | pSB1AK3 consisting BBa_B0014 | P1T1_for | Term_rev | 63 | 116 + | 15 |
Table 1: This table shows the three different Phusion PCR reactions that were carried out today. If this is successful, these three parts (part 1, 2 and 4) along with (part 3) which is already obtained can be ligated together for the construction of subtilin immunity BioBrick with the help of Gibson Cloning method.
- The extension rate of the Phusion polymerase is 1 Kb/ 30 seconds. Therefore the extension time of each PCR reaction is different.
- The primers in bold are the new primers we received today. The primers not in bold were previously re-hydrated.
Discussion and Conclusion
Gel electrophoresis will be carried out tomorrow, 20th August, 2010 to check for the amplified spaIFEG gene cluster, Promoter and RBS (pVeg-SpoVG) and Double terminator.
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