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| Line 3: |
Line 3: |
| | =pGA Vector Assay= | | =pGA Vector Assay= |
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| - | 1. Prepare stocks for protease inhibitor, DNAse, and lysozyme. | + | 1. |
| - | *a. Protease Inhibitor = 25x stock ; one tablet into 2ml of buffer
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| - | *b. DNAses = 100x stock (10mg/ml initial → 0.1mg/mL final)
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| - | *c. Lysozyme = 10x stock (10mg/ml initial → 1mg/mL final)
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| - | 2. Resuspend 50ml of each type of cell culture in 1ml of this mixture: 850ul sodium phosphate buffer, 40ul protease inhibitor, 10ul DNAse (0.1mg/ml final), and 100ul lysozyme (1mg/ml final).If testing multiple reactions, just multiply the volumes and divide into eppendorf tubes so that each reaction gets 1mL of cell suspension.
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| - | *a. Buffer: 1ml = 1000ul - (40+10+100) → 850ul
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| - | *b. Protease inhibitor: 25x (xml) = 1x (1ml) → 40ul
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| - | *c. DNAses: (10mg/ml)Xul = (0.1mg/ml)1ml → 10ul
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| - | *d. Lysozyme: (1mg/ml)Xul = (1mg/ml) 1ml → 100ul
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| - | 3. To lyse cells, add 111uL 10% triton (1% final) and mix the tube at room temperature for 30 min. Then, centrifuge the tubes at max. speed for 15 min (or until the liquid is very clear and not cloudy.
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| - | *x/(1000 +x) = 1/10 ….................. x = 111ul
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| - | 4. Transfer the liquid on top to new labeled eppendorf tube (leave the pellet of lysed stuff; our protein should be sufficiently soluble in aqueous solutions), tube perhaps containing master mix to initiate reaction.
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| - | 5. Place Target polyspring inserts into the Target DP glass vials.
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| - | 6. Cell Lysis
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| - | *a. Sonicate --use micro tip, 60%, 30 on 30 off (POST-assay: cell lysate not so effective)
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| - | *b. Bugbuster --Use 10x bugbuster, follow protocol (POST-assay: cell lysate not so effective)
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| - | *c. Triton X-100 --Use 1% triton (POST-assay: most effective for cell lysate)
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