Part:BBa_K1431101:Design

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.

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:
   
   Thermocycling Conditions for a Routine PCR:
   
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:
   
7. Ligase vector and insert at 16℃ for 45min and inactive at 60℃ for 10min. Ligation protocol shows:
   
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.