User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/09/06

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Confirm The BSMB1 Removal

Two seperate bands in each column show the vector backbone and the insert, 4442 bps and the BD001 (KAH201+KAH182) (1901) constructed gene.Backbone, 1901 bps, Cut with SpeI and NotI. First column: Ladder, columns 1-5 are 3:1 ratio colonies, column6 is 4:1 ratio, column 7 is 5:1 ratio and column 8 is 10:1
Plasmid Type SD-BD002 1 SD-BD002 SD-BD002 BD002
Plasmid DNA 3.0 μl 3.0 μl 3.0 μl 3.0 μl
BSMB1 1.0 μl 0.0 μL 0.0 μL 1.0 μL
Spe1 0.0 μl 1.0 μL 0.0 μL 1.0μL
10x FastDigest buffer + green loading dye 1.5 μl 1.5 μl 1.5 μl 1.5 μl
dH2O 9.5 μl 9.5 μL 10.5 μL 9.5 μL
  15.0 μl total 15μL 15μL 15μL
Incubate at 37°C for 10 minutes.

1- SD-BD002 means BD002 plasmid after the Site-Directed Mutagenesis.

  1. Make the (1%) agarose glee and add 15μl of restricted vector in one well and 10 μl of ladder.