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

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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/(X/P)/203 + (2) KAH182/(S/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 (X/P) TBD TBD TBD
2. KAH182 (S/P) TBD TBD TBD


> Ligations (edit after ligation is done)

Ligation Plate results (lig : neg crtl) 11/31/12
1. KAH201 (X/P)/203, x ng + KAH182 (S/P)/1675, x ng ---
2. KAH182 (S/P)/ 25 ng ---
  1 2
Insert DNA --- ---
Vector DNA --- ---
2x lgn buf (Roche) --- ---
T4 ligase (NEB) --- ---
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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