From 2011.igem.org

pGA Vector Assay

1. Prepare stocks for protease inhibitor, DNAse, and lysozyme.

• a. Protease Inhibitor = 25x stock ; one tablet into 2ml of buffer
• b. DNAses = 100x stock (10mg/ml initial → 0.1mg/mL final)
• c. Lysozyme = 10x stock (10mg/ml initial → 1mg/mL final)

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.

• a. Buffer: 1ml = 1000ul - (40+10+100) → 850ul
• b. Protease inhibitor: 25x (xml) = 1x (1ml) → 40ul
• c. DNAses: (10mg/ml)Xul = (0.1mg/ml)1ml → 10ul
• d. Lysozyme: (1mg/ml)Xul = (1mg/ml) 1ml → 100ul

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.

• x/(1000 +x) = 1/10 ….................. x = 111ul

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. 5. Place Target polyspring inserts into the Target DP glass vials. 6. Cell Lysis

• a. Sonicate --use micro tip, 60%, 30 on 30 off (POST-assay: cell lysate not so effective)
• b. Bugbuster --Use 10x bugbuster, follow protocol (POST-assay: cell lysate not so effective)
• c. Triton X-100 --Use 1% triton (POST-assay: most effective for cell lysate)