Difference between revisions of "Part:BBa K174000:Design"
 
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498 bp long sspB CDS is amplified by PCR with dangling end primers with EcoRI-NotI-XbaI restriction site at 5’ and SpeI at 3’ and inserted into a Biobrick compatible vector. The sequence is taken from E. coli strain DH5 alpha. Genbank accession number for E. coli MG1655 strain is NC_000913.2
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The sequence is taken from E. coli strain DH5 alpha. Genbank accession number for E. coli MG1655 strain is NC_000913.2
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Forward primer used: GATCTG-GAATTCGCGGCCGCTTCTAG-ATGGATTTGTCACAGCTAAC
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                    (Clamp sequence - Standard Biobrick prefix - first 20 base from the Biobrick) 
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Reverse primer used: TGTGAC-ACTAGTA-TTACTTCACAACGCGTAATGC
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                    (Clamp sequence - SpeI site - last 21 base from the Biobrick)
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==Construction==
 
==Construction==
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Treat Plasmid part BBa_J04450 (pSB1AT3) and our construct with EcoRI and SpeI
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Plasmid part BBa_J04450 (pSB1AT3) and our construct were treated with EcoRI and SpeI
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[[Image:Newcastle_2009_sspB_1.png‎]]
 
[[Image:Newcastle_2009_sspB_1.png‎]]
  
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Ligation of the backbone and the sspB insert
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The backbone and the sspB insert were then ligated
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[[Image:Newcastle_2009_sspB_2.png‎]]
 
[[Image:Newcastle_2009_sspB_2.png‎]]

Latest revision as of 22:36, 21 October 2009

The sequence is taken from E. coli strain DH5 alpha. Genbank accession number for E. coli MG1655 strain is NC_000913.2

Construction

Plasmid part BBa_J04450 (pSB1AT3) and our construct were treated with EcoRI and SpeI

Newcastle 2009 sspB 1.png

The backbone and the sspB insert were then ligated

Newcastle 2009 sspB 2.png