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Team:Newcastle/19 August 2010
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==Materials and protocol== | ==Materials and protocol== | ||
| - | Please refer to [[Team:Newcastle/DNA_re-hydration| DNA rehydration]] page for the protocol for the rehydration of the four primers and to the [[Team:Newcastle/PCR| Phusion PCR]] page for protocol followed for Phusion PCR | + | Please refer to [[Team:Newcastle/DNA_re-hydration| DNA rehydration]] page for the protocol for the rehydration of the four primers and to the [[Team:Newcastle/PCR| Phusion PCR]] page for protocol followed for Phusion PCR. |
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'''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. | '''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. | + | *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. | *The primers in bold are the '''new primers''' we received today. The primers not in bold were previously re-hydrated. | ||
Revision as of 02:20, 26 October 2010
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Contents |
Gel extraction and ligation of yneA into pGFPrrnB and PSB1AT3
Aims
The aim of the experiment is to further purify the yneA, pGFPrrnB and PSB1AT3 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.
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
Tommorrow we will carry out a test gel to check the sizes of the amplified fragments and if the sizes are correct then gel extraction will be performed
rocF BioBrick
Gel extraction of linearised pSB1C3
Aims
We digested the plasmid pSB1C3 with the restriction enzyme EcoR1 to linearize it and then we would be running the sequence on the gel to check if the plasmid has become linear or not and then we would be extracting it.
Materials and protocol
Please refer to the:
- gel electrophoresis,
- gel extraction and
- NanoDrop spectrophotometer protocols.
Results
- Lane 1: 1 Kb ladder
- Lane 2: Linearized plasmid pSB1C3
- Lane 3: 1 Kb ladder
There is no gel photograph because we want to keep the exposure of DNA to the UV light to an absolute minimum.
Discussion
During gel extraction procedure, we found a bright band of approximately 3100 bp size in lane 2 under UV light and we cut the gel and extracted the band.
Conclusion
We got linearized plasmid pSB1C3 and we performed gel extraction successfully and the nanodrop protocol showed that we got 22 ng/µl concentration of plasmid.
PCR of pSB1C3, pspac oid and double terminator with new primers
Aim
The aim of this experiment is to amplify plasmid linearized pSB1C3, Pspac_oid promoter and double terminator for the construction of rocF BioBrick with the help of 3 different Phusion PCR.
Materials and Protocol
Please refer to PCR for Phusion PCR protocol. The details for the 3 PCR reactions are mentioned below:
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 | P1V1 forward | P2V1 reverse | 65 | 2072 approx. | 65 |
| 2 | Pspacoid Promoter | pMutin4 | Prom_forward | P2P1 reverse | 67 | 106 approx. | 15 |
| 3 | Double Terminator | pSB1AK3 consisting BBa_B0014 biobrick | P1T1 forward | Term_reverse | 63 | 116 approx. | 15 |
Table 2: Table represents 3 different Phusion PCR reactions for the amplification of linearized plasmid pSB1C3, Pspac_oid promoter and double treminator so that it can be ligated together with other fragments for the construction of rocF with the help of Gibson Cloning method.
- The extension rate of the Phusion polymerase is 1Kb/ 30 seconds. Thus the extension time of each and every PCR reaction is slightly different.
- For more about the rocF fragments, please refer to the Cloning strategy for rocF.
Discussion
All the 3 Phusion PCR reactions were done however, gel electrophoresis will be done tomorrow, to check whether the fragments have actually amplified or not.
Conclusion
Tomorrow we will be running gel electrophoresis to check the outcome of the 3 PCR reactions and later all the fragments will be ligated with help of Gibson protocol if the fragments have amplified.
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