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Team:Newcastle/19 August 2010
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| - | '''Table | + | '''Table #''': This table shows the four different Phusion PCR reactions that were carried out today. If this is successful, the four parts 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. Thus the extension time of each and every PCR reaction is slightly different. | * The extension rate of the Phusion polymerase is 1 Kb/ 30 seconds. Thus the extension time of each and every PCR reaction is slightly different. | ||
* Please note: The newly arrived primer tubes have to be handled with extra caution, because they will be the main stocks which working stock solutions will be made from. Therefore, gloves have to be worn, as well as preventing any contamination. Water will be used in order to liquefy the primers and the water used will be from Pure Lab Distilled Water. | * Please note: The newly arrived primer tubes have to be handled with extra caution, because they will be the main stocks which working stock solutions will be made from. Therefore, gloves have to be worn, as well as preventing any contamination. Water will be used in order to liquefy the primers and the water used will be from Pure Lab Distilled Water. | ||
Revision as of 08:30, 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 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 #: This table shows the four different Phusion PCR reactions that were carried out today. If this is successful, the four parts 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. Thus the extension time of each and every PCR reaction is slightly different.
- Please note: The newly arrived primer tubes have to be handled with extra caution, because they will be the main stocks which working stock solutions will be made from. Therefore, gloves have to be worn, as well as preventing any contamination. Water will be used in order to liquefy the primers and the water used will be from Pure Lab Distilled Water.
rocF BioBrick
Gel extraction of linearised pSB1C3
PCR of pSB1C3, pspac oid and double terminator with new primers
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