User:Robwarden/Notebook/Divalent Ligand Validation/2009/03/14

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Make New Amp Plates

  • Amp100
  • Spit Test

Ligation

Reactions

  • pMSCVN (XhoI & NotI) + H1C (XhoI & NotI)
  • pMSCVN (XhoI & NotI) + H1Y (XhoI & NotI)
  • pMSCVN (XhoI & NotI) + Water (Negative Control)

Setup

9.4 μL H1C/H1Y/Water digest
0.6 μL pMSCVN digest
 10 μL Quick Ligation Buffer
  1 μL Quick Ligase

Conditions

Incubate 5 min at room temperature.

Transformation

Reaction

  50 μL XL10-Blue Supercompetent Cells
0.85 μL beta-mercaptoethanol
   5 μL Ligation Product

Conditions

  1. Ice for 30 min.
  2. 42°C Water bath for 45 sec.
  3. Ice for 2 min. Add 450μL preheated SOC.
  4. Shake at 37°C for 1 hr.
  5. Plate 150mL on each plate.
  6. Incubate at 37°C overnight.