User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/04/22

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Date

Building a Reporter Gene to Determine Chromatin Protein Function (Ligation into the mammalian vector)


  • Ligation of construct KAH182-KAH201 and mammalian vector V0200 and cloning in DH5α-T.
  • KAH182 = BBa_S04739 and KAH201 = BBa_S04745 and V0200 = BBa_J176121.
  • Assembly strategy: The backbone V0200(N/S)/4442 , the insert is KAH182-KAH201(N/S)/1901.


Digests


(1) KAH182-KAH201, 1901 bp, (2) V0200, 4442 bp, Both 1 and 2 are cut with Spe1 and Not11
Plasmid DNA 25.00 μl
NotI 1.0 μl
SpeI 1.0 μl
10x FastDigest buffer + green loading dye 3.00 μl
dH2O 0.00 μl
  30.0 μl total
Incubate at 37°C for 10 minutes.
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