User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2012/11/20

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11/20/12


Assemblies: Shown as Part/cuts/purified fragment size

  1. KAH109/pT7CFE1-CHis: KAH109/(E/P)/1132 + pT7CFE1-CHis/(E/P)/3600
  2. KAH111/pT7CFE1-CHis: KAH184/(E/S)/1123 + "
  3. KAH112/pT7CFE1-CHis: KAH187/(E/S)/1123 + "
  4. KAH115/pT7CFE1-CHis: KAH188/(E/S)/931 + "
  5. KAH148/pT7CFE1-CHis: KAH189/(E/S)/967 + "
  6. KAH150/pT7CFE1-CHis: KAH190/(E/S)/967 + "
  7. KAH151/pT7CFE1-CHis: KAH191/(E/S)/967 + "
  • Note: KAH109 is being re-done as an internal control to trouble-shoot the ligation
  • Note: KAH111-KAH151 are all cut and purified already


Digest (Fermentas FD)

  1. pT7CFE1-CHis, E/P
  2. KAH109, E/P
  3. KAH111, E/P
  4. KAH112, E/P
  5. KAH115, E/P
  6. KAH148, E/P
  7. KAH150, E/P
  8. KAH151, E/P
Reagent Volume  
DNA (plasmid) 25.0 μL
10x buffer 3.0
EcoRI 1.0
PstI 1.0
dH2O 0
  30 μL --> 37°C/ ~30 min.


Measure concentration(s)

Sample OD260 260/280 ng/μL
1. pT7CFE1-CHis (E/P) 57.344 1.829 ---
2. KAH109, (E/P) --- 36.072 2.354
3. KAH111, (E/P) --- 15.003 2.238
4. KAH112, (E/P) --- 22.857 1.862
5. KAH115, (E/P) --- 18.312 2.096
6. KAH148, (E/P) --- 23.735 2.389
7. KAH150, (E/P) --- 19.822 1.99
8. KAH151, (E/P) --- 19.701 1.90



Ligations (trouble shooting suggestions)

  • Note: Use 50 ng of vector this time (instead of 20)
  • Note: Use a 3:1 ratio of insert to vector... x μL insert = 3 * (bp of insert/ bp of vector) * 50 ng vector / (concentration of insert ng/μL)
  1. BD-116 (KAH10
  1 2 3 4 5 6 7 8
Inser Name KAH109 KAH111 KAH112 KAH115 KAH148 KAH150 KAH151 N/A
Insert DNA 1.3 3.12 2.05 2.12 1.7 2.03 2.05 0
Vector DNA 0.87 0.87 0.87 0.87 0.87 0.87 0.87 0.87
2x lgn buf (Roche) 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
T4 ligase (NEB) 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
dH2O 1.83 0.01 1.08 1.01 1.43 1.1 1.08 3.13
  10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL 10 μL


  • Add total ligation reaction to 30 μL DH5α-Turbo cells
  • Follow routine "fast" transformation procedure
  • Plate onto 100 ug/mL Amp agar plates