User:Karmella Haynes/Notebook/BioBrick cloning/2013/03/14: Difference between revisions
From OpenWetWare
(8 intermediate revisions by the same user not shown) | |||
Line 8: | Line 8: | ||
==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 | |||
Revision as of 13:45, 9 April 2013
Karmella's BioBrick Cloning | <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page <html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html> </html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html> |
04/14/13
New mammalian expression vector MV9
|