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=== | ||
| − | Design the primers and PCR by | + | Design the primers and PCR by Q5<sup>®</sup> High-Fidelity DNA Polymerases from plasmid. |
| − | Primer Forward: T TCTAGA TGGGATCAAGACTGGACAAGA<br> | + | |
| − | Primer Reverse: AAAACTGCAG CGGCCGC T ACTAGT A CCGAAGCCCAACCTTTCATA<br> | + | <center>https://static.igem.org/mediawiki/parts/9/9b/Plasmid_transfected.jpg</center> |
| + | <center>'''pBX-093 PB5-HS4-SV40-puro-2A-tetON3G-pA-HS4-TRE-AzaminGreen-2A-T'''</center><br> | ||
| + | |||
| + | |||
| + | ===Protocol=== | ||
| + | #Design the primers show below:<br>Primer Forward: T TCTAGA TGGGATCAAGACTGGACAAGA<br>Primer Reverse: AAAACTGCAG CGGCCGC T ACTAGT A CCGAAGCCCAACCTTTCATA<br> | ||
| + | #Dissolve the primers into 50pmol/μl | ||
| + | #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 | ||
| + | #Purify by Gel Purification Kit | ||
| + | #Using Nanodrop to get each sample’s concentration | ||
| + | #Digest by XbaI and PstI overnight. The digest protocol shows below:<br>https://static.igem.org/mediawiki/parts/e/ec/SUSTC_digest_system.png | ||
| + | #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 | ||
| + | #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:<br> | ||
| + | VF2: tgccacctgacgtctaagaa<br> | ||
| + | VR: attaccgcctttgagtgagc | ||
| + | |||
| + | The result shows the same sequence with our ideal design. | ||
===Source=== | ===Source=== | ||
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:
- 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.