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Team:Newcastle/Labwork/24 September 2009
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Formal Lab Session - 24th September 2009
Overview
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.
Sporulation Tuning/Chassis Team
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.
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
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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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