Difference between revisions of "Part:BBa K3407008:Design"

 
 
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===Design Notes===
 
===Design Notes===
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The target sequence (protospacer) is a sequence of 20 nucleotides that must precede a PAM sequence (NGG).
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The biobrick is constructed by two PCR’s on a commercially bought plasmid pKDsgRNA-p15 and a Gibson Assembly. The primers used were the following:
  
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1rst PCR:
  
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forward primer 5’ -  tttataacctccttagagctcga - 3’
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reverse primer 5’ - <b>tgcatccactaaagttaccg </b>gttttagagctagaaatagcaag - 3’
  
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===Source===
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Second PCR:
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forward primer 5’ - ccaattgtccatattgcatca 3’
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reverse primer 5’ - <b>cggtaactttagtggatgcag</b>gtgctcagtatctctatcactga - 3’.
  
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Synthetically synthesised
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The sequence of the protospacer is highlighted in bold.
  
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===References===
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A Gibson Assembly was done with both PCR fragments and the resulting plasmid was transformed in <i>E. coli</i> DH5alpha and recovered on LB with 60 ng/µl spectinomycin. The plasmid was confirmed by sequencing.

Latest revision as of 21:24, 27 October 2020


sgRNA targetting 4.3 gene of T7 bacteriophage


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]


Design Notes

The biobrick is constructed by two PCR’s on a commercially bought plasmid pKDsgRNA-p15 and a Gibson Assembly. The primers used were the following:

1rst PCR: 

forward primer 5’ -  tttataacctccttagagctcga - 3’ 
reverse primer 5’ - tgcatccactaaagttaccg gttttagagctagaaatagcaag - 3’

Second PCR: 

forward primer 5’ - ccaattgtccatattgcatca 3’ 
reverse primer 5’ - cggtaactttagtggatgcaggtgctcagtatctctatcactga - 3’.

The sequence of the protospacer is highlighted in bold.

A Gibson Assembly was done with both PCR fragments and the resulting plasmid was transformed in E. coli DH5alpha and recovered on LB with 60 ng/µl spectinomycin. The plasmid was confirmed by sequencing.