IGEM:Imperial/2010/Vectors team: Difference between revisions

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[[Image:AmyE_assembly_Stategy.JPG |750px|thumb|center|alt=A|Overveiw of AmyE assembly]]
[[Image:AmyE_assembly_Stategy.JPG |750px|thumb|center|alt=A|Overveiw of AmyE assembly]]
Starting from the top, we are assembling the first two fragments (K14070 and K14064) and K147002 with our oligos to add in a Dif site. Two dif sites on either side of resistance cassettes can be used to later excise antibiotic resistance from our final constructs.
[[Image:K70_n_K64.JPG |250px|thumb|right|alt=A|A : K143070 and K11064 assembly step]]
* K14070 and K14064 fragments
- DNA was taken out of the reigstry
- Cut with restriction enzymes
- Run on a gel to confirm correct cutting
  and estimate relative ratios of DNA for ligation
- Ligated overnight
- Transformed into E.Coli to Amplify the DNA
- Colony PCR has been used to confirm the correct insert size.
* Next Steps:
- Extract the DNA with a miniprep
- Proceed to the next step - Reverse PCR
[[Image:K02_and_oligos.JPG |250px|thumb|right|alt=A|B : K14002 and oligo assembly]]
*K14002 and oligos
- Ligated two single stranded oligos together to produce Dif and insertion site
- Standard biobrick assembly of oligos to K14002
- Ligation and transformation into E.coli competent cells (strain)
- Screen plated colonies for correct insertion
*Next Steps
- Purify the correct insert out of E.coli
- Next step assembly - LacI test vector and Final assembly vector
[[Image:LacI_testing_construct |250px|thumb|right|alt=A|B2 : LacI Testing Vector]]
*LacI Testing vector
- Currently waiting for Part B step 2 Midi-prep results to start this step

Latest revision as of 12:30, 12 September 2010

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Objectives

We are assembling the AmyE and PyrD vectors in order to transform B. Subtilis with our parts. Once completed, these vectors will be reusable and can then be used to introduce any relevant piece of DNA directly into B.subtilis genome. The vectors will be used both for the final assembly and for testing constructs.

Assembly of Vectors

A
Overveiw of PyrD assembly



A
Overveiw of AmyE assembly
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