Haynes Lab:Notebook/Engineering PC-TFs/2012/04/02

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Summary

  • Assembly 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### 1.3 0.4 0.5 0.3 -----------
DNA Vector(flyPCD) 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
  • 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 failed. TROUBLESHOOTING: Never ever ever put stock enzymes on the heat block. Stock enzymes should always be kept on ice or in the white & green cooling block. Should pre-warm the agar plates for about 30 minutes before you plate the cells. With zero colonies, probably forgot to add something to ligation reactions.