Difference between revisions of "Part:BBa K300001:Design"

 
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<partinfo>BBa_K300001 short</partinfo>
 
<partinfo>BBa_K300001 short</partinfo>
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===Source===
 
===Source===
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#<partinfo>BBa_K300980</partinfo>, provided by Mr Gene DNA synthesis service (www.mrgene.com), was excised from its original shipping vector (pMA) through digestion with MfeI (Fermentas) and NsiI (Fermentas) restriction enzymes. It was then isolated through a 1% agarose gel electrophoresis and gel-extracted (Macherey-Nagel NucleoSpin Extract II).
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#<partinfo>BBa_I763007</partinfo>, available in the Registry, was digested with EcoRI-PstI (Roche), gel-ran and its <partinfo>pSB1A2</partinfo> vector was gel-extracted as described above.
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#Digested <partinfo>BBa_K300980</partinfo> and <partinfo>pSB1A2</partinfo> have compatible ends (EcoRI-MfeI and PstI-NsiI). They were ligated with T4 Ligase (Roche) and transformed into competent TOP10 E. coli strain (<partinfo>BBa_V1009</partinfo>) which were then plated on Ampicillin (100 ug/ml) plates.
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#Positive transformants were identified by colony PCR with VF2 (<partinfo>BBa_G00100</partinfo>) and VR (<partinfo>BBa_G00101</partinfo>) standard primers and by restriction mapping with EcoRI (Roche) or NsiI (Fermentas). The yielded plasmid was <partinfo>BBa_K300001</partinfo>-<partinfo>BBa_B0033</partinfo> (i.e. there is the <partinfo>BBa_B0033</partinfo> RBS in the vector cloning site).
  
 
===References===
 
===References===

Revision as of 01:20, 10 October 2010

BioBrick integrative base vector for S. cerevisiae


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Plasmid lacks a suffix.
    Illegal XbaI site found at 46
    Illegal SpeI site found at 2
    Illegal PstI site found at 261
    Illegal PstI site found at 1607
    Illegal PstI site found at 3663
  • 12
    INCOMPATIBLE WITH RFC[12]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 3727
    Illegal NheI site found at 1549
    Illegal NheI site found at 1600
    Illegal SpeI site found at 2
    Illegal PstI site found at 261
    Illegal PstI site found at 1607
    Illegal PstI site found at 3663
    Illegal NotI site found at 3733
  • 21
    INCOMPATIBLE WITH RFC[21]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal EcoRI site found at 3727
    Illegal BglII site found at 51
    Illegal XhoI site found at 1499
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found at 3727
    Plasmid lacks a suffix.
    Illegal XbaI site found at 46
    Illegal SpeI site found at 2
    Illegal PstI site found at 261
    Illegal PstI site found at 1607
    Illegal PstI site found at 3663
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found at 3727
    Plasmid lacks a suffix.
    Illegal XbaI site found at 46
    Illegal XbaI site found at 3742
    Illegal SpeI site found at 2
    Illegal PstI site found at 261
    Illegal PstI site found at 1607
    Illegal PstI site found at 3663
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Plasmid lacks a prefix.
    Plasmid lacks a suffix.
    Illegal BsaI.rc site found at 2695


Design Notes

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Source

  1. BBa_K300980, provided by Mr Gene DNA synthesis service (www.mrgene.com), was excised from its original shipping vector (pMA) through digestion with MfeI (Fermentas) and NsiI (Fermentas) restriction enzymes. It was then isolated through a 1% agarose gel electrophoresis and gel-extracted (Macherey-Nagel NucleoSpin Extract II).
  2. BBa_I763007, available in the Registry, was digested with EcoRI-PstI (Roche), gel-ran and its pSB1A2 vector was gel-extracted as described above.
  3. Digested BBa_K300980 and pSB1A2 have compatible ends (EcoRI-MfeI and PstI-NsiI). They were ligated with T4 Ligase (Roche) and transformed into competent TOP10 E. coli strain (BBa_V1009) which were then plated on Ampicillin (100 ug/ml) plates.
  4. Positive transformants were identified by colony PCR with VF2 (BBa_G00100) and VR (BBa_G00101) standard primers and by restriction mapping with EcoRI (Roche) or NsiI (Fermentas). The yielded plasmid was BBa_K300001-BBa_B0033 (i.e. there is the BBa_B0033 RBS in the vector cloning site).

References