User:Karmella Haynes/Notebook/BioBrick cloning/2013/03/14: Difference between revisions
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|style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span> | |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]]<span style="font-size:22px;">Karmella's BioBrick Cloning</span> | ||
|style="background-color: #F2F2F2" align="center"| | |style="background-color: #F2F2F2" align="center"|[[File:Report.png|frameless|link={{#sub:{{FULLPAGENAME}}|0|-11}}]][[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]<br />{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[File:Resultset_previous.png|frameless|link={{#lnpreventry:{{FULLPAGENAME}}}}]][[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]] }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]][[File:Resultset_next.png|frameless|link={{#lnnextentry:{{FULLPAGENAME}}}}]]}} | ||
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==04/14/13== | ==04/14/13== | ||
<!-- Precede finished items with a checkmark ✓ --> | <!-- Precede finished items with a checkmark ✓ --> | ||
* New | * New vector: design new vector for mammalian expression | ||
* Order oligos | * Order oligos | ||
---- | ---- | ||
'''New mammalian expression | '''New mammalian expression vector MV9'''<br> | ||
* Use the [http://gcat.davidson.edu/igem10/ Oligator tool] to design oligo-assembly inserts for "MV6" (pcDNA3.1+ puro, mammalian transfection vector; CMV promoter) | * Use the [http://gcat.davidson.edu/igem10/ Oligator tool] to design oligo-assembly inserts for "MV6" (pcDNA3.1+ puro, mammalian transfection vector; CMV promoter) | ||
* MV9 - will carry Kozak-XbaI-NLS-6His-stop (for Brady's and Behzad's projects) | |||
** | ** The XbaI site will be used to accept X/S inserts; plasmid digests will be needed to check proper orientation | ||
* Oligo assembly method will be used to make double stranded DNA with SpeI overhangs | * Oligo assembly method will be used to make double stranded DNA with SpeI overhangs | ||
* | * Insert will be ligated to XbaI-cut MV6 to destroy the preexisting XbaI site | ||
* Clones will be screened for proper orientation and insert copy number | * Clones will be screened for proper orientation and insert copy number | ||
Oligator results: | Oligator results: | ||
* Custom prefix top = ctagt | |||
* Custom prefix bottom = a | |||
* Custom suffix top = a | |||
* Custom suffix bottom = tgatc | |||
<font face="courier"> | |||
<u>ctagtCCCGCCGCCACCATGGAGTCTAGAC</u>CCAAGAAAAAGCGCAAGGTACACCATCACCACCATCACGCGTAAAGCTGAGa<br> | |||
aGGGCGGCGGTGGTACCTCAGATCTGGGTTCTTTTTCGCGTTCCATGTGGTA<u>GTGGTGGTAGTGCGCATTTCGACTCtgatc</u></font><br> | |||
# 30-mer 5'-ctagtCCCGCCGCCACCATGGAGTCTAGAC | |||
# 52-mer 5'-CCAAGAAAAAGCGCAAGGTACACCATCACCACCATCACGCGTAAAGCTGAGa | |||
# 52-mer 5'-ATGGTGTACCTTGCGCTTTTTCTTGGGTCTAGACTCCATGGTGGCGGCGGGa | |||
# 30-mer 5'-ctagtCTCAGCTTTACGCGTGATGGTGGTG | |||
Latest revision as of 22:32, 26 September 2017
Karmella's BioBrick Cloning | Main project page Previous entry Next entry |
04/14/13
New mammalian expression vector MV9
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