Difference between revisions of "Part:BBa K1668007:Design"
 
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===Source===
 
===Source===
  
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The CDS<i>plu1537</i> gene was amplified by PCR with the template genomic DNA extracted from strain <i>Photorhabdus luminescens TT01</i>. We got the strain from Shandong University.
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The CDS<i>plu1537</i> gene was amplified by PCR with the template genomic DNA extracted from strain <i>Photorhabdus luminescens</i> TT01. We got the strain from Shandong University.
 
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<h4>PCR</h4>
 
<h4>PCR</h4>
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The CDS<i>plu1537</i> gene was amplified by PCR with the template genomic DNA extracted from strain <i>Photorhabdus luminescens TT01</i>.  
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The CDS<i>plu1537</i> gene was amplified by PCR with the template genomic DNA extracted from strain <i>Photorhabdus luminescens </i>TT01.  
 
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plu1537 F(F, 5’-3’): AATTCGCGGCCGCTTCTAGATGTCAGAGATCGAAGTAAT <br><br>
 
plu1537 F(F, 5’-3’): AATTCGCGGCCGCTTCTAGATGTCAGAGATCGAAGTAAT <br><br>
 
plu1537 R(R, 5’-3’): CGGACTGCAGCGGCCGCTACTAGTATTATTAGCTTACAATCATATATTC <br><br>
 
plu1537 R(R, 5’-3’): CGGACTGCAGCGGCCGCTACTAGTATTATTAGCTTACAATCATATATTC <br><br>
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<h4>Transformation and confirmation</h4>
 
<h4>Transformation and confirmation</h4>
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After seamless assembly, standard plasmid pSB1C3 containing <i>plu1537</i> gene was transformed into <i> E.coli DH5α</i>. When single colony appeared on the LB plate, we picked out 10 colonies, respectively, as our template for bacteria solution PCR. In order to avoid the appearance of false positive clones, we used VF2/VR as the universal primers. The positive clone and its corresponding raw bacteria solution were stored and samples were sent to do DNA sequencing.
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After seamless assembly, standard plasmid pSB1C3 containing <i>plu1537</i> gene was transformed into <i> E.coli</i> DH5α. When single colony appeared on the LB plate, we picked out 10 colonies, respectively, as our template for bacteria solution PCR. In order to avoid the appearance of false positive clones, we used VF2/VR as the universal primers. The positive clone and its corresponding raw bacteria solution were stored and samples were sent to do DNA sequencing.
 
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<h4>Plasmid map</h4>
 
<h4>Plasmid map</h4>
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[[File:ZJU-CHINA_TPCDS_1537.PNG|300px|thumb|left|Fig.3 the plasmid map of BBa_K1668007]]
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[[File:ZJU-CHINA_TPCDS_1537.PNG|300px|thumb|left|Figure 4 the plasmid map of BBa_K1668007]]
 
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Latest revision as of 10:44, 18 September 2015

CDS plu1537


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]



Source

The CDSplu1537 gene was amplified by PCR with the template genomic DNA extracted from strain Photorhabdus luminescens TT01. We got the strain from Shandong University.

Design Notes

PCR

The CDSplu1537 gene was amplified by PCR with the template genomic DNA extracted from strain Photorhabdus luminescens TT01.

We use primer plu1537-left F and plu1537-left R to amplify the left side of gene, which are shown below.

Seamless assembly

We used seamless assembly as our assembly method so restriction digestion and T4 ligation can be avoided. Detailed protocol and instruction for primer design can be seen in our Protocol. By this way prefix sequence, plu1537 and suffix sequence can be ligated seamlessly.

plu1537 F(F, 5’-3’): AATTCGCGGCCGCTTCTAGATGTCAGAGATCGAAGTAAT

plu1537 R(R, 5’-3’): CGGACTGCAGCGGCCGCTACTAGTATTATTAGCTTACAATCATATATTC


Transformation and confirmation

After seamless assembly, standard plasmid pSB1C3 containing plu1537 gene was transformed into E.coli DH5α. When single colony appeared on the LB plate, we picked out 10 colonies, respectively, as our template for bacteria solution PCR. In order to avoid the appearance of false positive clones, we used VF2/VR as the universal primers. The positive clone and its corresponding raw bacteria solution were stored and samples were sent to do DNA sequencing.

Plasmid map

Figure 4 the plasmid map of BBa_K1668007





















References