Haynes Lab:Notebook/Engineering PC-TFs/2012/04/05

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4/5/12

  • Ligation Scheme

Number beside vector and insert is number of base pairs

  • 1. flyPCD (186) + KAH204 (2328)
  • 2. flyPCD (186) + KAH205 (1251)
  • 3. flyPCD (186) + KAH206 (1551)
  • 4. flyPCD (186) + KAH225 (1251)
  • 5. flyPCD (186) only
' 1 2 3 4 5
DNA Insert KAH###1.30.40.50.3-----------
DNA Vector(flyPCD)0.30.30.30.30.3
T4 Ligase11111
Lign Buffer (2x)55555
dH2O2.43.33.23.43.6
10 µL10 µL10 µL10 µL10 µL
  • In a .5 mL tube, pipette the ligation buffer first.
  • Then water, DNA insert, vector, and T4 ligase.
  • Once completed, allow them to incubate for 10 minutes.
  • Meanwhile, get ice bucket and allow competent E. coli cells to thaw for five minutes.
  • Then follow transformation process:


Transformation Process
  • Warm five 100 μg/mL Amp agar plates at 37 °C
  • Thaw fresh tube of DH5α Turbo cells on ice
  • Include #5 tube, water only, no DNA (negative control)
  • Incubate cells + DNA on ice for 5 min.
  • Label pre-warmed plates
  • Transfer cells + DNA onto agar
  • Add 10 - 15 sterile glass beads, shake, discard beads
  • Incubate plates at 37 °C overnight
Ligation Results
Ligation Results

10:1 ratio with negative control

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