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Team:Newcastle/10 August 2010
From 2010.igem.org
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Gel extraction of rocF BioBrick components
Gel Electrophoresis for Amplified Pspac_oid promoter, Double terminator and Plasmid Vector pSB1C3
Aim
The aim of the experiment is to perform gel electrophoresis for the amplified fragments of Pspac_oid promoter, double terminator and plasmid vector pSB1C3 and thus perform gel exraction of the required bands.
Materials and Protocol
Please refer to: Gel electrophoresis.
Result
The experiment is divided in two separate gels. For plasmid vector pSB1C3, we used 1% agarose gel but for Psapc_oid and double terminator fragment we used 1.5% agarose gel for a better resolution. We used 1Kb DNA ladder (Promega) for the gel containing plasmid vector while we used 100bp DNA ladder (Promega) for the gel containing Pspac_oid and double terminator fragment.
| Pspac_oid pormoter | Double Terminator | Plasmid Vector pSB1C3 | |
|---|---|---|---|
| Size of the Fragment (in bp) | 148 approx. | 116 approx. | 2072 approx. |
Table 1: Table represents the sizes of the Pspac_oid fragment, Double terminator and plasmid vector pSB1C3 represented as bands on the gel in their respective lanes.
Discussion
We found bands of appropriate sizes in their respective lanes.
Conclusion
Gel electrophoresis was successful and now we would be doing gel extraction of all the six fragments (including the three fragments on which we did gel electrophoresis today) fragments.
Gel extraction of all 6 fragments for rocF
Aim
The aim of the experiment is to perform gel electrophoresis for the amplified fragments of Pspac_oid promoter, double terminator and plasmid vector pSB1C3 and thus perform gel exraction of the required bands.
Materials and Protocol
Please refer to: Gel extraction.
Result
The experiment is divided in two separate gels. For plasmid vector pSB1C3, we used 1% agarose gel but for Psapc_oid and double terminator fragment we used 1.5% agarose gel for a better resolution. We used 1Kb DNA ladder (Promega) for the gel containing plasmid vector while we used 100bp DNA ladder (Promega) for the gel containing Pspac_oid and double terminator fragment.
| Pspac_oid pormoter | Double Terminator | Plasmid Vector pSB1C3 | |
|---|---|---|---|
| Size of the Fragment (in bp) | 148 approx. | 116 approx. | 2072 approx. |
Table 1: Table represents the sizes of the Pspac_oid fragment, Double terminator and plasmid vector pSB1C3 represented as bands on the gel in their respective lanes.
Discussion
We found bands of appropriate sizes in their respective lanes.
Conclusion
Gel electrophoresis was successful and now we would be doing gel extraction of all the six fragments (including the three fragments on which we did gel electrophoresis today) fragments.
Gibson assembly of rocF BioBrick
lacI and pVeg
Aims
We aim to extract pVeg and lacI from transformed E.coli DH5α which was cultured in LB broth overnight(see 09.08.2010)check the concentration of DNA using nanodrop and perform a restriction digest so we can run the samples on a gel to check the insert sizes.
Materials and Protocols
| Restriction digest EcoR1 and Pst1 | ||||
| DNA | EcoR1 | Pst1 | 10 times buffer | Water |
|---|---|---|---|---|
| 5 | 0.5 | 0.5 | 1 | 3 |
See protocols for materials
Results
Results from Nanodrop:
| lacI 1 | lacI 2 | lacI 3 | lacI 4 | pVeg | |
|---|---|---|---|---|---|
| Concentration of DNA ng/microlitre | 122.5 | 139.4 | 130.0 | 150.0 | 155.0 |
Subtilin Immunity BioBrick
Aims
Subtilin is required as a cell-signalling molecule in order to trigger calcium carbonate precipitation, as well as bacteria filament formation. We aim to produce 2 subtilin BioBricks; Production and Immunity. Because subtilin is a lantibiotic that is found naturally in Bacillus subtilis ATCC 6633, the introduction of subtilin into our Bacillus 168 cells would actually kill them. Therefore, it is essential to establish immunity for 168 strains.
Lab work today will involve the rehydration of the primers that has arrived to us. These primers are Primer 1 P-1, Primer 2 P-2, Primer 1 S-1, Primer 2 S-1, Primer 1 T-1 and Primer 2 T-1. The other primers are Primer 1 V-1 and Primer 2 V-1, which we already had in stock, therefore no rehydration will be needed. Primer 1 = Forward primer and Primer 2 = Reverse primer.
These 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.
Materials and Protocol
Please refer to the Phusion PCR protocol.
   
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