拟南芥原生质体亚细胞定位Copy of Localization of procedure.docxVIP

亚细胞定位 Fresh Arabidopsis leaves Fast temp to 4oC for the centrifugation rotor Thaw the enzyme solution in ice and put the w5 solution in the 4oc freezer 准备冰块于冰盒 准备各种试剂 （黄色部分），需要准备的仪器等（红色部分）; Freshly PEG buffer need prepare before isolation of proplast because need 30 min to cool down to room temperature. I.Prepare for proplasts All steps carry out at 4oC or keep ice cold. The upper epidermal surface was stabilized by Time tape（一般的胶带子即可）, then use scissor to trim the remaining no use part of tape, while the lower epidermal surface was affixed to a strip of Magic tape（这个较重要，家乐福可买到）. The Magic tape was then carefully pulled away from the Time tape. 2-4 big, fresh and mature leaves were used. The peeled leaves which still adhering to the Time tape, were transferred to a Petri dish(就是一次性的皿，毛金成有) containing 10-15mL of enzyme solution. The leaves were gently shaker (40rpm on a platform shaker，速度不能过快) under darkness for about 2 hr until the protoplasts were released into the solution. Pour （枪吸易破） the solution into 50mL tube and then centrifuge 60 g for 5min（需要提前冷却到四度）, washed twice (40g, 4min) with 25 mL of pre-chilled modified w5 solution （可将此液先行倒在50mL tube于冰中） and incubated on ice for 30min (First, use 1mL to easily resuspend and then add other 24mL solution, and then up and down gently). Note：For three samples, pour about 20mL W5 into 50mL tube and keep it at RT for next procedure; every sample needs at least 1.5mL *3 + 500uL during (II) step. During this period, protoplasts were counted using a hemocytometer under a 20X light microscope. No. of protoplast in 25mL solution= No. of total five square of intact protoplasts *(25/5)*104 = 15*5*104=7.5*105 this time. 需要准备计数板 The protoplasts were then centrifuged and resuspended in modified MMg solution to final concentration of 10 *105 cells/mL. (in this time thus need to use 750uL MMg solution to resuspend proplasts) II.Proplast transfection assays All steps carry out at RT; its function just like the heat induction to make