Difference between revisions of "Part:BBa K2446041:Design"

 
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===Design Notes===
 
===Design Notes===
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This part was a fusion protein which assembled by three fragments OE-PCR. It contain three core domains from N-terminal to C-terminal: ZF_43-8 DNA binding domain (DBD), nuclear location sequence and transcription regulating domain.
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<B>OE-PCR:</B> PCR amplify and connecting the necessary fragments, using proofreading polymerase enzyme. They should have about 15-25 bp overlaps. Use oligo Tm calculators to figure out their annealing temperature. Clean up or gel extract the correct size band. Use cleaned up fragments as "template". Unlike normal PCR, about 1/2 to 3/4 volume of the extension reaction should be template.
  
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1.Use proofreading enzyme for extension.
  
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2.Run 10-15 PCR cycles without end primers. (Template extension step)
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3.Add end primers, then continue cycling for another 15-20 rounds.
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4.Gel extract the correct fragment. Clone into a vector.
  
 
===Source===
 
===Source===

Revision as of 07:44, 31 October 2017


ZF_43-8_KRAB


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 301
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 184
    Illegal AgeI site found at 19
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This part was a fusion protein which assembled by three fragments OE-PCR. It contain three core domains from N-terminal to C-terminal: ZF_43-8 DNA binding domain (DBD), nuclear location sequence and transcription regulating domain.

OE-PCR: PCR amplify and connecting the necessary fragments, using proofreading polymerase enzyme. They should have about 15-25 bp overlaps. Use oligo Tm calculators to figure out their annealing temperature. Clean up or gel extract the correct size band. Use cleaned up fragments as "template". Unlike normal PCR, about 1/2 to 3/4 volume of the extension reaction should be template.

1.Use proofreading enzyme for extension.

2.Run 10-15 PCR cycles without end primers. (Template extension step)

3.Add end primers, then continue cycling for another 15-20 rounds.

4.Gel extract the correct fragment. Clone into a vector.

Source

waiting

===References===