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| ==04/14/13== | | ==04/14/13== |
| <!-- Precede finished items with a checkmark ✓ --> | | <!-- Precede finished items with a checkmark ✓ --> |
| * New vectors: design new vectors for mammalian expression | | * New vector: design new vector for mammalian expression |
| * Order oligos | | * Order oligos |
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| ---- | | ---- |
| '''New mammalian expression vectors MV9 and MV10'''<br> | | '''New mammalian expression vector MV9 and MV10'''<br> |
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| * 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 (Brady's and Behzad's project)
| | * MV9 - will carry Kozak-XbaI-NLS-6His-stop (for Brady's and Behzad's projects) |
| ** MV10 - will carry Kozak-XbaI-stop
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| * 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 |
| * Inserts will be ligated to XbaI cut MV6 to destroy the preexisting XbaI site | | * 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 |
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| # 30-mer 5'-CTAGTCTCAGCTTTACGCGTGATGGTGGTG | | # 30-mer 5'-CTAGTCTCAGCTTTACGCGTGATGGTGGTG |
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| '''Kozak-XbaI-stop'''<br>
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| ----
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| '''Assemblies'''
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| # BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
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| # BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
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| * Digests (Fermentas FD)
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| ** Specific notes
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| {| {{table}} cellspacing="3" <!-- Digest rxn. table -->
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| |- valign="top"
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| | bgcolor=#cfcfcf | Reagent
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| | bgcolor=#cfcfcf | Volume
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| | rowspan="7" | <!-- [[Image:GelImage.jpg|270px|Hover name]]<br>30 μL/lane, 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
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| | DNA (plasmid) || up to 25 μL
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| | 10x buffer || 3.0
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| |-
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| | enzyme 1 || 1.0
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| | enzyme 2 || 1.0
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| | dH<sub>2</sub>O || ---
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| | || 30 μL --> 37°C/ ~30 min.
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| |}
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| * Measure conc.'s
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| {| {{table}} cellspacing="3" <!-- [DNA] table -->
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| |- bgcolor=#cfcfcf
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| | Sample || OD260 || 260/280 || ng/μL
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| | 1. Digested part (a/b) || --- || --- || ---
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| |-
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| | 2. Digested part (c/d) || --- || --- || ---
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| |}
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| * Dephosphorylation (Roche)
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| {| {{table}} cellspacing="3" <!-- Dephos table -->
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| |-
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| | bgcolor=#cfcfcf | Reagent
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| | bgcolor=#cfcfcf | Volume
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| | DNA (clean digest) || up to 17 μL (500 ng)
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| | 10x buffer d.p. || 2.0
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| | phosphatase || 1.0
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| | dH<sub>2</sub>O || ---
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| | || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
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| |}
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| * Ligations
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| {| {{table}} cellspacing="3" <!-- Ligations table -->
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| |- bgcolor=#cfcfcf
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| | Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
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| | 1. insert(a/b)/size, ## ng + vector(c/d)/size, ## ng || <font color="blue">new BioBrick #:1 (Pick #)</font>
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| | 2. vector(c/d)/ ## ng ||
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| |}
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| {| {{table}} cellspacing="3" <!-- Ligation rxn table -->
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| | || 1 || 2 ||
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| | Insert DNA || ### || --- ||
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| | Vector DNA || ### || ### ||
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| | 2x lgn buf (Roche) || ### || ### ||
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| | T4 ligase (NEB) || 1.0 || 1.0 ||
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| | dH<sub>2</sub>O || ### || ### ||
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| | || # μL || # μL ||
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| |}
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| ----
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| '''Oligo annealing'''
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| # New BB 1
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| # New BB 2
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| {| class="wikitable" border="0" cellspacing="3" <!-- Oligo annealing rxn table -->
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| | DNA (oligos, 100 μM) || up to 18 μL (3 μL ea.)
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| | 10x annealing buffer || 2.0
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| | dH<sub>2</sub>O || ---
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| | || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
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| |}
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