User:Rigzin N. Dekhang/Notebook/Nucleoprotein Comlex Assembly/Entry Base

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Vesicle Prep

(i) 1 litre yeast culture at 34 degree celsius, OD measured at 600 (should be 0.8-1) after that add 5 ml 1M NaN3 to each 1 litre flasks. Swirl it.
(ii) Decant 400 ml from each flask into a Nalgene tube ~ need 10 tubes.
(iii) Spin cells at 7,000 rpm for 15 mins (JA-10 rotor)
(iv) Discard the supernatant. Wash the cells with NaN3+H20 (5ml+950ml). Resuspend the pellets. After repeated washing, distribute it in 2 Nalgene tubes filled to the rim and spin the cells at 7,000 rpm for 15 minutes (JA-10). Precaution: Make sure weigh and balance the tubes before spinning)
(v) Discard the sup. Pretreat the cells with 250 ml of Pre-treatment buffer for each tube for 10 mins at RT.
(vi) Spin down the cells at 7,000 rpm for 15 minutes.
(viii) Discard the supernatant. Wash pellets with 100 ml of SWB for each tube.
(ix) Spin at 6,000 rpm for 10 minutes (JA-10 rotor).
(x) Resuspend the cells in 75 ml SWB for each tube. Add 200 mg zymolyase + 2.5 ml of glusulase. Divide it in 4X50 ml falcon tubes. (xi) Rotate at 34 degree for 2 hours.