Coding
Part:BBa_K1431101:Design
Designed by: Rifei Chen, Yushan Zhang Group: iGEM14_SUSTC-Shenzhen (2014-10-09)
TetOn-3G, an ideal controller of mammalian gene expression with TRE-3G promoter+PolyA
Assembly Compatibility:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
Design the primers and PCR by Q5® High-Fidelity DNA Polymerases from plasmid.
Protocol
- Design the primers show below:
Primer Forward: T TCTAGA TGGGATCAAGACTGGACAAGA
Primer Reverse: AAAACTGCAG CGGCCGC T ACTAGT A CCGAAGCCCAACCTTTCATA - Dissolve the primers into 50pmol/μl
- PCR by the protocol below:
Thermocycling Conditions for a Routine PCR:
- Purify by Gel Purification Kit
- Using Nanodrop to get each sample’s concentration
- Digest by XbaI and PstI overnight. The digest protocol shows below:
- Ligase vector and insert at 16℃ for 45min and inactive at 60℃ for 10min. Ligation protocol shows:
- 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.