User:Robwarden/Notebook/Divalent Ligand Validation/2009/02/25

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Colony Screen

Digestion

 10 μL DNA
  2 μL 10x NEB Buffer 3 ->   26 μL
0.2 μL 100x BSA         ->  2.6 μL
0.5 μL NotI             ->   13 μL
  1 μL NdeI             ->    6 μL
6.3 μL MilliQ           -> 81.9 μL

Results

All 12 colonies showed correct bands. Colony #6 was a little off. DNA from colonies 1-5,7-12 combined & quantified (179 ng/μL). LB added to liquid culture #5, grown overnight for glycerol stock.

Digestion/Phosphatase

Reaction

  15 μL pMSCVN (2.7 μg)
   5 μL 10X NEB Buffer 3
 0.5 μL 100X BSA
   2 μL XhoI
   2 μL NotI
25.5 μL MilliQ

Conditions

Incubate at 37°C for 3.5 hrs.

Inactivation

Incubate at 65°C for 30 min.

Phosphatase

Add 5μL 10X Antarctic Phosphatase Buffer and 1μL Antarctic Phosphatase. Incubate at 37°C for 1 hr. Inactivate at 65°C for 5 min.

Final Concentration

[pMSCVN] = 47.9 ng/μL

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 (1:10 dil'n)
 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.


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