Team:Newcastle/Labwork/24 September 2009

=Formal Lab Session - 24th September 2009= = Overview = 
 * Metal Sensor Team - 
 * Stochastic Switch Team - 
 * Sporulation Tuning/Chassis 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:



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

Introduction

 * 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.

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.