Difference between revisions of "Haynes Lab:Notebook/Engineering PC-TFs/2013/03/27"

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* Meanwhile, get ice bucket and allow competent E. coli cells to thaw for five minutes.
 
* Meanwhile, get ice bucket and allow competent E. coli cells to thaw for five minutes.
 
* Then follow transformation process:
 
* 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
  
  

Revision as of 08:08, 2 April 2013

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3/27/2013

  • 1. Kozak,V0120 + BL01
  • 2. Kozak,V0120 + BL05
  • 3. Kozak,V0120 + BL09
  • 4. Kozak,V0120 + BL12
1 2 3 4 5
DNA Insert 1.4 1.8 2.0 2.5 --
DNA Vector (Kozak) 0.6 0.6 0.6 0.6 0.6
T4 Ligase 1 1 1 1 1
Lign Buffer (2x) 5 5 5 5 5
dH2O 2.0 1.6 1.4 .9 3.7
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