Haynes Lab:Notebook/Engineering PC-TFs/2012/03/01

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Summary

Assembly Scheme: Number beside vector and insert is number of base pairs

  • 1. hPCD (186) + KAH204 (2328)
  • 2. hPCD (186) + KAH205 (1251)
  • 3. hPCD (186) + KAH206 (1551)
  • 4. hPCD (186) + KAH225 (1251)
  • 5. hPCD (186) only
' 1 2 3 4 5
DNA Insert 1.3 0.4 0.5 0.3 -----------
DNA Vector 0.3 0.3 0.3 0.3 0.3
T4 Ligase 1 1 1 1 1
Lign Buffer (2x) 5 5 5 5 5
dH2O 2.4 3.3 3.2 3.4 3.6
10 µL 10 µL 10 µL 10 µL 10 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
  • Add 30 μL of DH5α Turbo cells to DNA + dH2O
  • 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


Template Table

' 1 2 3 4 5
DNA Insert -- -- -- -- -----------
DNA Vector -- -- -- -- --
T4 Ligase 1 1 1 1 1
Lign Buffer (2x) 5 5 5 5 5
dH2O -- -- -- -- --
10 µL 10 µL 10 µL 10 µL 10 µL