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| | colspan="2"| | | | colspan="2"| |
| <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> | | <!-- ##### DO NOT edit above this line unless you know what you are doing. ##### --> |
| ==mm/dd/yy== | | ==04/18/13== |
| <!-- Precede finished items with a checkmark ✓ --> | | <!-- Precede finished items with a checkmark ✓ --> |
| * Line item 1 | | * MV9 cloning - sequencing order |
| * Line item 2
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| ---- | | ---- |
| '''Minipreps'''<br> | | '''MV9 sequencing'''<br> |
| * Check with E/P digests | | * DNASU |
| | | ** DNA: 200 ng/ 10 μL |
| {| {{table}} border="1" cellspacing="3" <!-- Digest check rxn. table -->
| | ** Primers: 16 pmol (16 μM) / 10 μL |
| |- valign="top"
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| | bgcolor=#cfcfcf | Reagent
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| | bgcolor=#cfcfcf | Volume
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| | rowspan="7" | <u>Expected:</u><br>1. BB 1 = size<br>2. BB2 = size<br>
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| | rowspan="7" | <!-- [[Image:GelImage.jpg|400px|Hover name]]<br>15 μL/lane; 1% agarose; [http://openwetware.org/wiki/Image:KAH_Fermentas_GeneRuler_1kbplus.jpg Ladder] -->
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| |-
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| | DNA(plasmid) || 2.0 μL
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| |-
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| | 10X buffer || 1.5
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| |-
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| | EcoRI || 1.0
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| |-
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| | PstI || 1.0
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| |-
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| | dH<sub>2</sub>O || 9.5
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| |-
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| | || 15 μL --> 37°C/ ~15 min.
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| |}
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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) | |
| ** 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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| |-
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| | 10x buffer || 3.0
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| |-
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| | enzyme 1 || 1.0
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| |-
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| | enzyme 2 || 1.0
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| |-
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| | dH<sub>2</sub>O || ---
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| |-
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| | || 30 μL --> 37°C/ ~30 min.
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| |}
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| | Sample || OD260 || 260/280 || ng/μL | | | Sample || OD260 || 260/280 || ng/μL |
| |- | | |- |
| | 1. Digested part (a/b) || --- || --- || --- | | | 1. 1-3 || 0.054 || 1.825 || 54.37 |
| |- | | |- |
| | 2. Digested part (c/d) || --- || --- || --- | | | 2. 3-5 || 0.047 || 1.73 || 47.14 |
| |}
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| * Dephosphorylation (Roche)
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| {| {{table}} cellspacing="3" <!-- Dephos table -->
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| |- | |
| | bgcolor=#cfcfcf | Reagent | |
| | bgcolor=#cfcfcf | Volume
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| |- | |
| | DNA (clean digest) || up to 17 μL (500 ng)
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| |-
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| | 10x buffer d.p. || 2.0
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| |- | | |- |
| | phosphatase || 1.0 | | | 3. 3-6 || 0.131 || 1.87 || 130.79 |
| |-
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| | dH<sub>2</sub>O || --- | |
| |- | |
| | || 20 μL --> 37°C/ 10 min.; 75°C/ 2 min.; [final] = 25 ng/μL
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| |} | | |} |
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| * Ligations | | * Sequencing results for order# 7177 |
| {| {{table}} cellspacing="3" <!-- Ligations table -->
| | ** MV9 1-3/ DD122B (fwd): correct sequence and orientation; reads through DD123B (rev primer) on other side |
| |- bgcolor=#cfcfcf
| | ** MV9 3-5/ DD122B (fwd): correct sequence and orientation; reads through DD123B (rev primer) on other side |
| | Ligation || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
| | ** MV9 3-6/ DD122B (fwd): correct sequence but backwards; reads through DD123B (rev primer) on other side |
| |-
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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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| |-
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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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| |-
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| | Insert DNA || ### || --- ||
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| |-
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| | Vector DNA || ### || ### ||
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| |-
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| | 2x lgn buf (Roche) || ### || ### ||
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| | T4 ligase (NEB) || 1.0 || 1.0 ||
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| |-
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| | dH<sub>2</sub>O || ### || ### ||
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| |-
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| | || # μL || # μL ||
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| |}
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| ---- | | * Keep MV9 1-3 in vector stocks. See [http://openwetware.org/wiki/User:Karmella_Haynes/Notebook/BioBrick_cloning/2013/04/12 colony PCR from 04/12/13] for gel confirmation. |
| '''Oligo annealing'''
| | * Keep MV9 3-6 in case it might be useful |
| # 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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| |-
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| | 10x annealing buffer || 2.0
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| |-
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| | dH<sub>2</sub>O || ---
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| |-
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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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