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Contents 1 Subtilin Immunity BioBrick 1.1 Aims 1.2 Materials and protocol 1.3 Results 1.4 Discussion 1.5 Conclusion

Subtilin Immunity BioBrick

Aims

We plan to select three spaIFEG gene (part 3) PCR tubes, using Tms 51°C, 56°C and 61°C (we had confirmed it successful from yesterday's gel electrophoresis - please refer to the gel electrophoresis from yesterday). Gel extraction will then be performed, along with Plasmid Vector (part 1), Promoter & RBS (part 2) and Double terminator (part 4). Finally, NanoDrop will be performed on each of these four parts.

Materials and protocol

Please refer to the gel electrophoresis, gel extraction and NanoDrop protocols. Instead of using the centrifuge to bind the DNA from our sample to the QIAquick column, a vacuum manifold was used instead. It is the first time it had been used. The advantage of this was that the extraction could be carried out much faster as we had 7 samples.

Results

The results from the Nanodrop Spectrophotometer of the four parts are as follows:

• Vector - 21.3 ng/μl
• Promoter - 28.8 ng/μl
• Coding sequence - 27.0 ng/μl
• Terminator - 27.5 ng/μl

Discussion

We chose the three spaIFEG gene (part 3) PCR tubes, using Tms 51°C, 56°C and 61°C because they were the mid-ranged Tms. Gel extraction was then performed, by firstly cutting the gels under U.V light, and then apply the protocol for completion.

Conclusion

The results from the NanoDrop reading suggests that our results were positive, which indicates that we have successfully extracted all the parts required for the Subtilin Immunity BioBrick. Gibson cloning method will be used next week.

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