Team:EPF-Lausanne/Notebook/Cloning Strategy

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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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=Cloning strategy=
 
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==July==
 
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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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===07.07.09===
 
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To design plasmids : software Vector NTI
 
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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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===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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===10.07.09===
 
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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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'''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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===14.07.09===
 
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[[Media: primer15+160709.pdf| Primers designed for LOVTAP read-out and RBphP project.]]
 
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===15.07.09===
 
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===16.07.09===
 
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===17.07.09===
 
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==August==
 
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</div><div CLASS="epfl09bouchon"></div>
 

Latest revision as of 08:05, 28 July 2009