User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/04/18

PcTF Subcloning in E. coli

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04/18/2013 Building a Reporter Gene to Determine Chromatin Protein Function Ligation of KAH182 = BBa_S04739 and KAH201 = BBa_S04745 and cloning in DH5α-T. Assembly strategy: The backbone KAH201(S/P)/3403 , the insert is KAH182(X/P)/1675. Digests (1,2) KAH182, 1675 bp, Cut with Xba1 and Pst1, (3,4) KAH201, 3403 bp, Cut with Spe1 and Pst1 Plasmid DNA 25.00 μl Enzyme 1* 1.0 μl Enzyme 2* 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. Enzyme 1 for KAH182 is Xba1 and for KAH201 is Spe1. Enzyme 2 for KAH182 and KAH201 is Pst1. Make the (1%) agarose glee and add 30μl of restricted vector in one well and 10 μl of ladder. Ligations Ligation 1. E. coli DH5α-T KAH182(X/P)/size, 15.85 ng + KAH201(S/P)/size, 36.29ng KAH182/KAH201 2:1 No Colonies 2. E. coli DH5α-T KAH182(X/P)/size, 15.85 ng + KAH201(S/P)/size, 36.29ng KAH182/KAH201 3:1 One Colony 3. E. coli DH5α-T KAH182(X/P)/size, 15.85 ng + KAH201(S/P)/size, 36.29ng KAH182/KAH201 4:1 Two Colonies 4. KAH201(S/P)/size, 36.29ng (Control Plate) No Colonies Calculations are for the ng of insert we need to get a 2:1, 3:1 and 4:1 ratios of insert molecules to 50 ng vector molecules   1 (2:1) 2 (3:1) 3 (4:1) 4 (- Ctrl) Insert DNA 3.11 4.66 6.21 --- Vector DNA 1.37 1.37 1.37 1.37 2x lgn buf (Roche) 5.48 7.03 8.58 5.0 T4 ligase (NEB) 1.0 1.0 1.0 1.0 dH2O 0.00 0.00 0.00 2.63   10.96 μL 14.06 μL 17.16μl 10μL The incubation time for the ligation process was 15min at room temperature. Fast transformation, 15 min on ice. Confirm The Assembly Two seperate bands in each column show the vector backbone and the insert, 4400 bps and the KAH201+KAH182 (226+1675) constructed gene.Backbone, 1901 bps, Cut with EcoRI and PstI. First column: Ladder, Second collumn: 3:1 ratio, Third and fourth column: 4:1 ratio (colony A and B) Plasmid DNA 2.0 μl EcoR1 1.0 μl Pst1 1.0 μl 10x FastDigest buffer + green loading dye 1.5 μl dH2O 9.5 μl   15.0 μl total Incubate at 37°C for 10 minutes. Make the (1%) agarose glee and add 15μl of restricted vector in one well and 10 μl of ladder.