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| - | {{EPF-Lausanne09}}
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| - | <div CLASS="epfltrick">__TOC__
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| - | </div><div CLASS="epfl09">
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| - |
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| - | =Cloning strategy=
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| - | ==July==
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| - |
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| - | ===06.07.09===
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| - | Four forward primers were designed to amplify:
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| - | <br> 1.Promoter T7, RBS, CBP and LOVTAP:
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| - | :gtttcttcgaattcgcggccgcttctagagtaatacgactcactataggggaattgtg
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| - | 2.RBS, CBP and LOVTAP:
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| - | :gtttcttcgaattcgcggccgcttctagagtgtttaactttaagaaggag
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| - | 3.CBP and LOVTAP:
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| - | :gtttcttcgaattcgcggccgcttctagatgaagcgacgatggaaaaagaatttcatag
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| - | 4.LOVTAP:
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| - | :gtttcttcgaattcgcggccgcttctagatgctactacacttgaacgtattgagaagaac
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| - | One reverse primer were designed:
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| - | :gtttcttcctgcagcggccgctactagtatcaatcgcttttcagcaacacctcttc
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| - | The '''recipient IGEM part''' have been chosen: [http://partsregistry.org/partsdb/get_part.cgi?part=BBa_B0010 BBa_B0010], well 13D in the received kit plate 1
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| - |
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| - | ===07.07.09===
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| - | To design plasmids : software Vector NTI
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| - |
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| - | ===08.07.09===
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| - | '''Inducible LOVTAP biobrick strategy'''
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| - | :*Problem to overcome:
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| - | :PstI sites in LOVTAP sequence.
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| - | :*Our goal:
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| - | :Biobrick consisted of LacI promoter-RBS-LOVTAP-Term (in this order).
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| - | :*Material:
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| - | :Biobrick of LacI promoter, RBS, LOVTAP, Term separately ( LOVTAP obtained from previous section and the rest from iGEM Spring 2009 distribution Kit plate).
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| - | :*Strategy:
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| - | :LacI promoter-RBS ligation with iGEM protocol (LacI promoter digested with ES, RBS digested with XP, plasmid containing an other antibiotic digested with EP).
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| - | :LOVTAP is digested with ES and inserted into Term plasmid (which was digested with EX previously).
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| - | :Finally, LacI promoter-RBS digested with ES to be inserted into LOVTAP-Term plasmid, digested with EX.
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| - |
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| - | ===09.07.09===
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| - | '''Partial digestion strategy'''
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| - | :* Problem to overcome:
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| - | :PstI sites in LOVTAP sequence -> Impossible to use iGEM protocol for ligation (where downstream part must be cut with XP)
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| - | :* Strategy:
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| - | :Cut with X first.
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| - | :Divide into several solutions and add different concentrations of P (by dilution series)
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| - | :*Results:
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| - | :Most concentrated tube: all P sites are recognized and cut, so the number of parts diminishes with the concentration value.
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| - | :Run an agarose gel and extract the right piece (recognized by the segment's length).
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| - |
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| - | ===10.07.09===
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| - |
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| - | ===13.07.09===
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| - | Restriction enzymes on [http://www.neb.com/nebecomm/products/category1.asp?#2 Biolabs website]
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| - | and [http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/cleavage_olignucleotides.asp clevage oligonucleotides]
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| - |
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| - | '''TRP promoter biobrick strategy'''
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| - | :* Problem to overcome:
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| - | : SpeI sites on Trp promoter sequence and it's an upstream part which has to be cut with ES.
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| - | :* Strategy:
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| - | : PCR: Forward primer having E and X sites and Reverse primer NheI.
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| - | : Digest Trp promoter with E and NheI.
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| - | : Digest plasmid with E and X.
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| - | : Ligation -> E site is recreated; X and NheI have compatible ends so ligation is possible and the site is destroyed (mixted site).
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| - |
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| - | ===14.07.09===
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| - | [[Media: primer15+160709.pdf| Primers designed for LOVTAP read-out and RBphP project.]]
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| - |
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| - | ===15.07.09===
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| - |
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| - | ===16.07.09===
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| - |
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| - | ===17.07.09===
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| - |
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| - | ===20.07.09===
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| - |
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| - | ===21.07.09===
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| - | One useful website to know the restriction sites of enzymes:
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| - | <br>http://www.genscript.com/cgi-bin/products/enzyme.cgi?op=all_ez
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| - | The restriction site used were:
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| - | <br>EcoRI GAATTC
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| - | <br>XbaI TCTAGA
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| - | <br>SpeI ACTAGT
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| - | <br>PsiI TTATAA
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| - | Design the primers for the 2 step-PCR:
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| - | the first step introduces the LacI promoter and the RBS upstream the LovTAP gene with the Forward primer, whereas the Reverse introduces the Term downstream.
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| - | In the second step we only introduce the E-X restriction sites upstream and the SP downstream.
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| - |
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| - | ===22.07.09===
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| - |
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| - | ===23.07.09===
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| - |
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| - | ===24.07.09===
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| - |
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| - | ===27.07.09===
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| - |
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| - | ===28.07.09===
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| - |
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| - | ===29.07.09===
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| - |
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| - | ===30.07.09===
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| - |
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| - | ===31.07.09===
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| - | ==August==
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| - |
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| - | </div><div CLASS="epfl09bouchon"></div>
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