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

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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.

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

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.

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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