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

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|              || 1    || 2    ||
|              || 1    || 2    ||
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|-
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| Insert DNA        || TBD || ---  ||
+
| Insert DNA        || 3.1 || ---  ||
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|-
-
| Vector DNA        || TBD || TBD ||
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| Vector DNA        || 0.13 || 0.13 ||
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| 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    || TBD || TBD ||
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| dH<sub>2</sub>O    || 0.77 || 3.87 ||
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| &nbsp;            || 10 μL || 10 μL ||
| &nbsp;            || 10 μL || 10 μL ||

Revision as of 16:26, 21 February 2013

Building a Reporter Gene Main project page
Previous entry      Next entry

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   Placeholder Image
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

Ligation Plate results (lig : neg crtl) mm/dd/yy
1. KAH201 (S/P)/203, 6 ng + KAH182 (X/P)/1675, 25 ng TBD
2. KAH182 (X/P)/1675 25 ng TBD
  1 2
Insert DNA 3.1 ---
Vector DNA 0.13 0.13
2x lgn buf (Roche) 5.0 5.0
T4 ligase (NEB) 1.0 1.0
dH2O 0.77 3.87
  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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