User:Karmella Haynes/Notebook/BioBrick cloning/2012/12/01

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(Autocreate 2012/12/01 Entry for User:Karmella_Haynes/Notebook/BioBrick_cloning)
(mm/dd/yy)
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==mm/dd/yy==
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==12/01/12==
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* Line item 1
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* Cultures/ minipreps: KAH3/MV2 colonies (2)
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* Line item 2
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----
----
'''Minipreps'''<br>
'''Minipreps'''<br>
-
> Check with E/P digests
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* Check with PstI digest
 +
* Compare to empty vector MV2
{| class="wikitable" border="0" cellspacing="3" <!-- Digest check rxn. table -->
{| class="wikitable" border="0" cellspacing="3" <!-- Digest check rxn. table -->
|-valign="top"
|-valign="top"
| <u>Reagent</u> || <u>Volume</u>  
| <u>Reagent</u> || <u>Volume</u>  
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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" | <u>Expected:</u><br>1. MV2 = 4529<br>2. KAH3/MV2-1 = 5280<br>3. KAH3/MV2-2 = 5280<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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| 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]
|-
|-
| DNA(plasmid) || 2.0 μL
| DNA(plasmid) || 2.0 μL
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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)<br>
 
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--> Specific notes<br>
 
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{| class="wikitable" border="0" cellspacing="3" <!-- Digest rxn. table -->
 
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|-valign="top"
 
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| <u>Reagent</u> || <u>Volume</u> || &nbsp;
 
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| rowspan="9" | <!-- [[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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|-
 
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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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| &nbsp; || 30 μL --> 37°C/ ~30 min.
 
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|}
 
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> Measure conc.'s
 
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{| class="wikitable" border="0" cellspacing="3" <!-- [DNA] table -->
 
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|-
 
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| <u>Sample</u> || <u>OD260</u> || <u>260/280</u> || <u>ng/μL</u>
 
-
|-
 
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| 1. Digested part (a/b) || --- || --- || ---
 
-
|-
 
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| 2. Digested part (c/d) || --- || --- || ---
 
-
|}
 
-
 
-
 
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> Dephosphorylation (Roche)
 
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{| class="wikitable" border="0" cellspacing="3" <!-- Dephos table -->
 
-
|-
 
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| <u>Reagent</u> || <u>Volume</u>
 
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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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{| class="wikitable" border="0" cellspacing="3" <!-- Ligations table -->
 
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| <u>Ligation</u> || <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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{| class="wikitable" border="0" 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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| 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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'''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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|}
 

Revision as of 17:19, 1 December 2012

Karmella's BioBrick Cloning Main project page
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12/01/12

  • Cultures/ minipreps: KAH3/MV2 colonies (2)



Minipreps

  • Check with PstI digest
  • Compare to empty vector MV2
Reagent Volume Expected:
1. MV2 = 4529
2. KAH3/MV2-1 = 5280
3. KAH3/MV2-2 = 5280
Image:GelImage.jpg
15 μL/lane; 1% agarose; Ladder
DNA(plasmid) 2.0 μL
10X buffer 1.5
EcoRI 1.0
PstI 1.0
dH2O 9.5
  15 μL --> 37°C/ ~15 min.



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