User:Karmella Haynes/Notebook/BioBrick cloning/2013/03/19

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(Autocreate 2013/03/19 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
Current revision (17:17, 22 March 2013) (view source)
(03/19/13)
 
(8 intermediate revisions not shown.)
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==mm/dd/yy==
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==03/19/13==
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* Line item 1
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* Oligo annealing for new mammalian vector
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* Line item 2
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----
 
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'''Minipreps'''<br>
 
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* Check with E/P digests
 
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{| {{table}} border="1" cellspacing="3" <!-- Digest check 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" | <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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| &nbsp; || 15 μL --> 37°C/ ~15 min.
 
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|}
 
----
----
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'''Assemblies'''
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'''Oligo annealing'''
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# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
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* Note: made fresh 10x annealing buffer
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# BioBrick name: 5' part/(a/b)/size + 3' part/(c/d)/size
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# KAH3/MV2(X) + K-XbaI-NLS-his-stop
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* K-XbaI-NLS-6his-st 1
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* K-XbaI-NLS-6his-st 2
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* K-XbaI-NLS-6his-st 3
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* K-XbaI-NLS-6his-st 4
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* Digests (Fermentas FD)
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{| {{table}} <!-- Oligo annealing rxn table -->
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** Specific notes
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| DNA (oligos, 100 μM) || 4x 3 μL ea. = 12.0
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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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|-
|-
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| DNA (plasmid) || up to 25 μL
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| 10x annealing buffer || 2.0
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|-
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| 10x buffer || 3.0
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| dH<sub>2</sub>O || 6.0
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|-
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| enzyme 1 || 1.0
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| &nbsp; || 20 μL  
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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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| &nbsp; || 30 μL --> 37°C/ ~30 min.
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|}
|}
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Thermal Cycling (try new thermal cycling approach)<br>
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* Measure conc.'s
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Multigene Optimax Thermal Cycler
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{| {{table}} cellspacing="3" <!-- [DNA] table -->
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* 99.9 °C, 10 min.
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|- bgcolor=#cfcfcf
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* [99.9°C, temp. increment -2.0°C, 1 min.; 90.0°C, temp. increment -2.0°C, 1 min.] x35
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| Sample || OD260 || 260/280 || ng/μL
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* 25°C, ∞
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|-
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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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|-
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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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|-
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| phosphatase || 1.0
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|-
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| dH<sub>2</sub>O || ---
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|-
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| &nbsp; || 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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|-
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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 || &nbsp;
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|}
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{| {{table}} cellspacing="3" <!-- Ligation rxn table -->
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| &nbsp;            || 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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|-
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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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| &nbsp;             || # 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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|-
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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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| &nbsp; || 20 μL --> 100°C (water bath)/ 5 min.; Cool to R.T. overnight
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|}
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Current revision

Karmella's BioBrick Cloning Main project page
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03/19/13

  • Oligo annealing for new mammalian vector



Oligo annealing

  • Note: made fresh 10x annealing buffer
  1. KAH3/MV2(X) + K-XbaI-NLS-his-stop
  • K-XbaI-NLS-6his-st 1
  • K-XbaI-NLS-6his-st 2
  • K-XbaI-NLS-6his-st 3
  • K-XbaI-NLS-6his-st 4
DNA (oligos, 100 μM) 4x 3 μL ea. = 12.0
10x annealing buffer 2.0
dH2O 6.0
  20 μL

Thermal Cycling (try new thermal cycling approach)
Multigene Optimax Thermal Cycler

  • 99.9 °C, 10 min.
  • [99.9°C, temp. increment -2.0°C, 1 min.; 90.0°C, temp. increment -2.0°C, 1 min.] x35
  • 25°C, ∞



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