User:Wesley J. Houston/Notebook/MAP/2013/02/08

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(February 8, 2013)
(February 8, 2013)
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> Ligations (edit after ligation is done)
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> Ligations
{| class="wikitable" border="0" cellspacing="3" <!-- Ligations table -->
{| class="wikitable" border="0" cellspacing="3" <!-- Ligations table -->
| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
| <u>Ligation</u> || <font color="blue"><u>Plate results (lig : neg crtl)</u> mm/dd/yy</font>
|-
|-
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| 1. KAH201 (S/P)/203, X ng + KAH182 (X/P)/1675, X ng || <font color="blue">---</font>
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| 1. KAH201 (S/P)/203, 6 ng + KAH182 (X/P)/1675, 25 ng || <font color="blue">---</font>
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|-
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| 2. KAH182 (X/P)/1685 X ng || ---
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| 2. KAH182 (X/P)/1685 25 ng || ---
|}
|}
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| &nbsp;            || 1    || 2    ||
| &nbsp;            || 1    || 2    ||
|-
|-
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| Insert DNA        || 0.1 || ---  ||
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| Insert DNA        || --- || ---  ||
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|-
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| Vector DNA        || 0.8 || 0.8 ||
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| Vector DNA        || --- || --- ||
|-
|-
| 2x lgn buf (Roche) || 5.0  || 5.0  ||
| 2x lgn buf (Roche) || 5.0  || 5.0  ||
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| T4 ligase (NEB)    || 1.0  || 1.0  ||
| T4 ligase (NEB)    || 1.0  || 1.0  ||
|-
|-
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| dH<sub>2</sub>O    || 3.1 || 3.2 ||
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| dH<sub>2</sub>O    || --- || --- ||
|-
|-
| &nbsp;            || 10 μL || 10 μL ||
| &nbsp;            || 10 μL || 10 μL ||

Revision as of 02:35, 7 February 2013

Building a Reporter Gene Main project page
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February 8, 2013

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



Assemblies

  1. KAH201/KAH182: (1) KAH201/(S/P)/203 + (2) KAH182/(X/P)/1675

> Digests (Fermentas FD)


Reagent Volume   Hover name
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 15 to 25 μL
10x buffer 3.0
enzyme 1 1.0
enzyme 2 1.0
dH2O 0 to 10 μL
  30 μL --> 37°C/ ~15 min.


> Measure conc.'s of gel purified fragments

Sample OD260 260/280 ng/μL
1. KAH201 (S/P) TBD TBD TBD
2. KAH182 (X/P) TBD TBD TBD


> Ligations

Ligation Plate results (lig : neg crtl) mm/dd/yy
1. KAH201 (S/P)/203, 6 ng + KAH182 (X/P)/1675, 25 ng ---
2. KAH182 (X/P)/1685 25 ng ---
  1 2
Insert DNA --- ---
Vector DNA --- ---
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O --- ---
  10 μL 10 μL
  • Follow quick transformation protocol
  • Add 30 uL DH5α-Turbo to each ligation; plate on 100 μg/mL Amp agar



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