User:Wesley J. Houston/Notebook/MAP/2013/03/04

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Current revision (18:56, 7 March 2013) (view source)
(3/4/13)
 
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==3/4/13==
==3/4/13==
*Second ligation was successful.
*Second ligation was successful.
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* Assembly: (5xGal4 : [Spacer : HSVtk TATA]) : (K : AmCyan : AmCyan : NLS : STOP : [polyA2]) (KAH201)/(KAH182); AmCyan reporter gene for chromatin proteins.
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----
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'''Assemblies'''
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* Note, KAH201 is in vector V0120, so add 3200 bp to the size.
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# KAH201/KAH182: (1) KAH201/(S/P)/203+3200 + (2) KAH182/(X/P)/1675
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> Digests (Fermentas FD)<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:Electrophoresis1.jpg‎ |150px|Results of the electrophoresis]]<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) || 20 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 || 5.0 μL
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|-
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| &nbsp; || 30 μL --> 37°C/ ~15 min.
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|}
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> Measure conc.'s of gel purified fragments
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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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|-
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| 1. KAH201 (S/P) || 0.048 || 1.88 || 48.1
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|-
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| 2. KAH182 (X/P) || 0.008 || 1.93 || 8.1
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|}
> Ligations (corrected version, 3/04/13)
> Ligations (corrected version, 3/04/13)

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3/4/13

  • Second ligation was successful.
  • Assembly: (5xGal4 : [Spacer : HSVtk TATA]) : (K : AmCyan : AmCyan : NLS : STOP : [polyA2]) (KAH201)/(KAH182); AmCyan reporter gene for chromatin proteins.



Assemblies

  • Note, KAH201 is in vector V0120, so add 3200 bp to the size.
  1. KAH201/KAH182: (1) KAH201/(S/P)/203+3200 + (2) KAH182/(X/P)/1675

> Digests (Fermentas FD)


Reagent Volume   Results of the electrophoresis
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 20 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O 5.0 μL
  30 μL --> 37°C/ ~15 min.


> Measure conc.'s of gel purified fragments

Sample OD260 260/280 ng/μL
1. KAH201 (S/P) 0.048 1.88 48.1
2. KAH182 (X/P) 0.008 1.93 8.1

> Ligations (corrected version, 3/04/13)

Ligation Plate results (lig : neg crtl) 03/04/13
1. KAH201 (S/P)/3403, 25 ng + KAH182 (X/P)/1675, 26 ng 10
2. KAH201 (S/P)/3403, 25 ng 0



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