Team:Newcastle/11 August 2010

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Gibson cloning of the rocF BioBrick

Aim

The aim of this experiment is to assemble the fragments we extracted yesterday in order to construct the rocF BioBrick and clone it into pSB1C3 in a single step isothermal reaction.

Materials and Protocol

Please refer to: Gibson cloning for materials required and the protocol for the reaction.

Result

The final result about the success of the Gibson reaction will be found on 13th August, 2010.

Discussion

We followed the whole procedure perfectly and later today after the reaction is complete, we would be transforming E. coli DH5α cells. We would be setting up 48 hours culture and by 13th August, 2010 we would be getting the result about the ligation of all 6 fragments of rocF.

Conclusion

The procedure went successfully and the result will be out by the end of 13th August, 2010.

Transformation of E. coli DH5α cells with ligated rocF fragments

Subtilin Immunity BioBrick

Aims

Our aims for today are to run the gel electrophoresis to check whether we have the correct fragement sizes on the four parts that are we amplified yesterday. If the PCR worked, we will then perform gel extraction and then perform another gel electrophoresis for the extracted gel in order to obtain our BioBrick parts.

Newcastle Loaded Gel for Subtilin Immunity BioBrick.JPG

Materials and protocol

Please refer to the gel electrophoresis and the gel extraction protocols.

Results

Newcastle Subtilin Immunity Gel 1.jpg

Figure #: Gel electrophoresis of the PCR products of the parts required for the subtilin immunity BioBrick.

  • Lane 1: 1 kb DNA ladder
  • Lane 2: Plasmid Vector (pSB1C3)
  • Lane 3: Promoter and RBS (pVeg-SpoVG)
  • Lane 4: spaIFEG Gene Cluster
  • Lane 5: Double terminator
  • Lane 6: 100 bp DNA ladder

Discussion

Plasmid Vector (lane 2), Promoter & RBS (lane 3) and Double terminator (lane 5) showed up. spaIFEG PCR tube (lane 4) did not show up. We think that this occurrence was due to the Tm error (it should be 63°C as opposed to 46°C). Therefore, another PCR and gel electrophoresis were performed.

Transformation of hyperspank and spoVG

Aim

To transform competent E. coli DH5α with hyperspank and spoVG.

Materials and Protocol

Please refer to transformation of E. coli.


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