Team:Newcastle/9 July 2010

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{{Team:Newcastle/mainbanner}}
{{Team:Newcastle/mainbanner}}
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==Transformation==
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==LacI BioBrick Construction==
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===Aims===
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=== Aims ===
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* To use PCR to extract ''lacI'' (promoter, ribosome-binding site (RBS) & coding sequence (CDS)) from plasmid pMutin4 and ligate into vector pSB1AT3 in front of red fluorescent protein (RFP).
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The aim of this experiment is to transform competent ''E. coli'' DH5α with pSB1AT3 vectors containing the lacI insert (in the ratios of 1:3 and 1:5 of vector to insert).
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===Materials===
===Materials===
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* Competent ''E. coli'' (DH5alpha strain)
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Each of the following five tubes contain 200 µl of competent ''E. coli'' DH5α. To this the DNA to be transformed was added.
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* Ligation of ''lacI'' into pSB1AT3
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# 1:3 (contains LacI insert and pSB1AT3 vector in the proportion of 1:3)
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# 1:5 (contains LacI insert and pSB1AT3 vector in the proportion of 1:5)
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# Negative control for ligation (contains vector with no insert)
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# Control for transformation (without plasmid)
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# Control for transformation (with plasmid, pSB1AT3)
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===Protocol===
===Protocol===
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* [[TeamNewcastleTransformation of E. coli|Transformation of ''E. coli'']] DH5alpha pSB1AT3 containing ''lacI'' insert.
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The [https://2010.igem.org/TeamNewcastleTransformation_of_E._coli#Protocol| transformation protocol] was followed using the tubes above:
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===Inference===
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*''Ecoli'' DH5alpha is used as a bioreactor to replicate our plasmids to create a large stock ready for extraction from cells.
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# Thaw a 200 µl aliquot of ''E. coli'' DH5α and add the transforming DNA. For tubes 1-3 5 µl of vector was added, for tube 4 no vector was added and for tube 5 only 1 µl of vector was added. This is because tubes 1 to 3 have been ligated and will contain cells in different forms of the ligated vector whereas tube 5 contains the ligated vector (our control).
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# Incubate for 45 mins on ice.
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# Heat-shock the cells at 42°C for 120 secs, and place on ice again for 3-4 min.
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# Add 1 ml of LB broth to each tube and incubate the cells at 37°C for 1.5 hr (static for initial 20 mins, shaking for remaining time).
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# Plate out 200 µl of solution from the 5 tubes above on LB plates (agar at 1.5%) using the glass balls - spread plating.
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#*11 LB plates were used (10 with ampicillin and 1 without ampicillin)
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#**3 plates for 1:3
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#**3 plates for 1:5
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#**2 vector only plates (with no insert) i.e. 1:0
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#**2 plates for without plasmid (one on LB with ampicillin and one without ampicillin)
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#**1 plate with pSB1AT3 plasmid (only 1 µl of plasmid purified by SW and DY (198 ng/µl) was added)
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# The plates were incubated overnight at 37°C and left in the fridge till Monday.
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===Results===
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Please see next day (link)
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{{Team:Newcastle/footer}}
{{Team:Newcastle/footer}}

Revision as of 12:24, 4 August 2010

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Contents

LacI BioBrick Construction

Aims

  • To use PCR to extract lacI (promoter, ribosome-binding site (RBS) & coding sequence (CDS)) from plasmid pMutin4 and ligate into vector pSB1AT3 in front of red fluorescent protein (RFP).

Materials

  • Competent E. coli (DH5alpha strain)
  • Ligation of lacI into pSB1AT3

Protocol

Inference

  • Ecoli DH5alpha is used as a bioreactor to replicate our plasmids to create a large stock ready for extraction from cells.
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