Difference between revisions of "Part:BBa K200005:Design"

(→‎Design Notes)
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===Design Notes===
 
===Design Notes===
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PCR from Escherichia Coli using BL21(DE3) as the genomic DNA template and the Pfu Ultra II enzyme. Primers were designed to include overhangs coding for XbaI and SpeI recognition sites in order to allow the gene to be BioBricked according to the BioBrick Standard. The primers are as follows:
  
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PCR from Escherichia Coli BL21(DE3) using Pfu Ultra II Polymerase.
 
  
−
 
+
Forward PCR primer containing XbaI overhang (bold) for BioBricking: <b>GCTCTAG</b>ATGAGTCGTTTAGTCGTAG      <br>
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Forward: <b>GCTCTAG</b>ATGAGTCGTTTAGTCGTAG      <br>
+
 
Recomended Temperatures for PCR : 52.4C(without overhang) and 63.2(with overhang)<br><br>
 
Recomended Temperatures for PCR : 52.4C(without overhang) and 63.2(with overhang)<br><br>
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Backward: <b>GGACTAGTA</b>CTACGCAAGCTTTGGAAAG      <br>
+
Reverse PCR primer containing SpeI overhang (bold) for BioBricking: <b>GGACTAGTA</b>CTACGCAAGCTTTGGAAAG      <br>
 
Recomended Temperatures for PCR : 54.5C(without overhang) and 65.1(with overhang)
 
Recomended Temperatures for PCR : 54.5C(without overhang) and 65.1(with overhang)
 
<br><br>
 
<br><br>

Latest revision as of 21:22, 19 October 2009

OtsA: Part 1 of 2 for trehalose producing enzymes.


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 383
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

PCR from Escherichia Coli using BL21(DE3) as the genomic DNA template and the Pfu Ultra II enzyme. Primers were designed to include overhangs coding for XbaI and SpeI recognition sites in order to allow the gene to be BioBricked according to the BioBrick Standard. The primers are as follows:


Forward PCR primer containing XbaI overhang (bold) for BioBricking: GCTCTAGATGAGTCGTTTAGTCGTAG
Recomended Temperatures for PCR : 52.4C(without overhang) and 63.2(with overhang)

Reverse PCR primer containing SpeI overhang (bold) for BioBricking: GGACTAGTACTACGCAAGCTTTGGAAAG
Recomended Temperatures for PCR : 54.5C(without overhang) and 65.1(with overhang)

BioBrick overhang shown in bold

Source

Escherichia coli BL21(DE3)

References