Team:Newcastle/Labwork/24 September 2009

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Team Newcastle 2009 iGEM ProbationaryP-Sign.PNG

Formal Lab Session - 24th September 2009

Team Newcastle 2009 iGEM 24-09-09 IMG 1379.JPG


Overview





Metal Sensor Team

To Do list

  1. PCR clean up of cotC PCR done on the 23/09/09
  2. Digest PCR clean up and also digest pMUTIN4 with BamHI and HindIII
  3. PCR clean up both of these products
  4. Run all of both sample on gel and excise bands
  5. Extract bands from gel using GenElute's Gel Extraction kit
  6. Set up overnight ligations of pMUTIN4 and cotC-GFP-smtA BioBrick

Stochastic Switch Team

Today we did minipreps of the ara/sspb/pSB1AT3 cultures which were then digested with EcoRI and SpeI. These were run on an 0.8% gel. We expected to see a ~600bp band as well as the plasmid backbone and the gel photograph seemed fairly convincing:

Team Newcastle240909 trans mini.png

We set up a 50ml midiprep culture from the culture corresponding to lane 9 which seemed to have the clearest band.

Sporulation Tuning/Chassis Team

Introduction

Team Newcastle 2009 iGEM 24-09-09 IMG 1373.JPG
  • We prepared kinA and pGFP-rrnB digested fragments yesterday, we'll carry on the next step as gel extraction and ligation and transformation.
  • For the double clone part, we need to ligate it to pMutin4 vector.


Experiment procedure

Gel extraction

  • Follow the standard procedure of Gel Extraction Kit(Sigma).

Ligation

  • Ligate kinA to pGFP-rrnB
  • Ligate digested PCR clean-up psc fragment to pMutin4
    • this psc fragment refer to the cwlJ:sleB fragment with HindIII and BamHI restriction sites at the end of each side.
 T4 ligase Buffer        2ul
 Vector                2.5ul
 insert DNA           14.5ul
 T4 ligase               1ul
 ---------------------------
                        20ul
  • -4C frige overnight.

Prepare the culture for Mini Prep

  • Since we got colonies from yesterday's transformation, we need to culture the colonies for mini prep.

Prepare smm medium

  • The protocal of making smm medium is come from our lab's B.subtilis 168 transformation protocal.

Conclusion

  • After the autoclave of smm medium, the medium got cloudy. It may has something wrong with the ingredients.
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