Difference between revisions of "Part:BBa K1431101:Design"

 
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<partinfo>BBa_K1431101 short</partinfo>
 
<partinfo>BBa_K1431101 short</partinfo>
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===Design Notes===
 
===Design Notes===
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It is useful for control reaction.
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Design the primers and PCR by Q5<sup>®</sup> High-Fidelity DNA Polymerases from plasmid.
  
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<center>https://static.igem.org/mediawiki/parts/9/9b/Plasmid_transfected.jpg</center>
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<center>'''pBX-093 PB5-HS4-SV40-puro-2A-tetON3G-pA-HS4-TRE-AzaminGreen-2A-T'''</center><br>
  
  
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===Source===
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===Protocol===
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#Design the primers show below:<br>Primer Forward: T TCTAGA TGGGATCAAGACTGGACAAGA<br>Primer Reverse: AAAACTGCAG CGGCCGC T ACTAGT A CCGAAGCCCAACCTTTCATA<br>
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#Dissolve the primers into 50pmol/μl
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#PCR by the protocol below:<br> https://static.igem.org/mediawiki/parts/c/cb/SUSTC_Q5_PCR_protocol.png<br>'''Thermocycling Conditions for a Routine PCR:'''<br>https://static.igem.org/mediawiki/parts/d/d2/SUSTC_Thermocycling_Conditions_for_a_Routine_PCR.png
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#Purify by Gel Purification Kit
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#Using Nanodrop to get each sample’s concentration
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#Digest by XbaI and PstI overnight. The digest protocol shows below:<br>https://static.igem.org/mediawiki/parts/e/ec/SUSTC_digest_system.png
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#Ligase vector and insert at 16℃ for 45min and inactive at 60℃ for 10min. Ligation protocol shows:<br>https://static.igem.org/mediawiki/parts/2/26/SUSTC_ligase.png
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#Transfect the plasmid into DH5α supercompetent cells
  
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The sequence we got it was from our instructor.
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===Sequencing Results===
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We sent fresh bacteria broth for sequencing using standard Biobricks sequencing primer VF2/VR. The sequencing cooperation we used is Invitrogen Guangzhou filiale.
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Sequence of sequencing primer we used:<br>
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VF2: tgccacctgacgtctaagaa<br>
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VR: attaccgcctttgagtgagc
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The result shows the same sequence with our ideal design.
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===Source===
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The plasmid was from our instructor, Huangwei's lab. And we design primers to copy down by PCR.
  
 
===References===
 
===References===

Latest revision as of 21:13, 26 October 2014

TetOn-3G, an ideal controller of mammalian gene expression with TRE-3G promoter+PolyA


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

Design the primers and PCR by Q5® High-Fidelity DNA Polymerases from plasmid.

Plasmid_transfected.jpg
pBX-093 PB5-HS4-SV40-puro-2A-tetON3G-pA-HS4-TRE-AzaminGreen-2A-T


Protocol

  1. Design the primers show below:
    Primer Forward: T TCTAGA TGGGATCAAGACTGGACAAGA
    Primer Reverse: AAAACTGCAG CGGCCGC T ACTAGT A CCGAAGCCCAACCTTTCATA
  2. Dissolve the primers into 50pmol/μl
  3. PCR by the protocol below:
    SUSTC_Q5_PCR_protocol.png
    Thermocycling Conditions for a Routine PCR:
    SUSTC_Thermocycling_Conditions_for_a_Routine_PCR.png
  4. Purify by Gel Purification Kit
  5. Using Nanodrop to get each sample’s concentration
  6. Digest by XbaI and PstI overnight. The digest protocol shows below:
    SUSTC_digest_system.png
  7. Ligase vector and insert at 16℃ for 45min and inactive at 60℃ for 10min. Ligation protocol shows:
    SUSTC_ligase.png
  8. Transfect the plasmid into DH5α supercompetent cells

Sequencing Results

We sent fresh bacteria broth for sequencing using standard Biobricks sequencing primer VF2/VR. The sequencing cooperation we used is Invitrogen Guangzhou filiale.

Sequence of sequencing primer we used:
VF2: tgccacctgacgtctaagaa
VR: attaccgcctttgagtgagc

The result shows the same sequence with our ideal design.

Source

The plasmid was from our instructor, Huangwei's lab. And we design primers to copy down by PCR.

References