Difference between revisions of "Part:BBa K191004:Design"

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===Design Notes===
 
===Design Notes===
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* Trp promoter was added to plasmid containing part <partinfo>BBa_I13507</partinfo> from iGEM kit using [http://2009.igem.org/wiki/index.php?title=Team:EPF-Lausanne/Protocols/Klenow Klenow protocol].
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* This BioBrick was synthesized using the protocol we developed with the Klenow fragment([http://2009.igem.org/wiki/index.php?title=Team:EPF-Lausanne/Protocols/Klenow complete protocol]).  
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* Trp promoter as PCR products in Klenow protocol was cut with E and S to be ligated into plasmid with RFP gene (<partinfo>BBa_I13507</partinfo>), which was also cut with E and S.
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* To do this, two long primers which together contained the sequence for the Trp promoter were ordered:
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Forward primer: 5'-gtttcttcgaattcgcggccgcttctagagtaatcatcgaactagttaactagtacgcaag-3'
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Reverse primer: 5'-gctagcgaacttgcgtactagttaactagttcgatg-3'
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* These primers could self-anneal in the reaction mix, and the second strands that were missing at the extremities of the primers were fully synthesized using the Klenow fragment, instead of a classic extension using the TAQ DNA Polymerase.
 +
* Double strand Trp promoter was cut with E and S to be ligated into plasmid with RFP gene (<partinfo>BBa_I13507</partinfo>).
  
 
===Source===
 
===Source===

Latest revision as of 16:26, 21 October 2009

TRP promoter - RBS - RFP - Term


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal SpeI site found at 43
    Illegal SpeI site found at 51
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal SpeI site found at 43
    Illegal SpeI site found at 51
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal SpeI site found at 43
    Illegal SpeI site found at 51
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal SpeI site found at 43
    Illegal SpeI site found at 51
    Illegal AgeI site found at 666
    Illegal AgeI site found at 778
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

  • This BioBrick was synthesized using the protocol we developed with the Klenow fragment([http://2009.igem.org/wiki/index.php?title=Team:EPF-Lausanne/Protocols/Klenow complete protocol]).
  • To do this, two long primers which together contained the sequence for the Trp promoter were ordered:

Forward primer: 5'-gtttcttcgaattcgcggccgcttctagagtaatcatcgaactagttaactagtacgcaag-3'

Reverse primer: 5'-gctagcgaacttgcgtactagttaactagttcgatg-3'

  • These primers could self-anneal in the reaction mix, and the second strands that were missing at the extremities of the primers were fully synthesized using the Klenow fragment, instead of a classic extension using the TAQ DNA Polymerase.
  • Double strand Trp promoter was cut with E and S to be ligated into plasmid with RFP gene (BBa_I13507).

Source

BBa_K191007, BBa_I13507

References