Coding

Part:BBa_K1431101:Design

Designed by: Rifei Chen, Yushan Zhang   Group: iGEM14_SUSTC-Shenzhen   (2014-10-09)
Revision as of 21:08, 26 October 2014 by ChenRifei (Talk | contribs) (Design Notes)

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.pngThermocycling 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