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nigel.lin銀蟲 (初入文壇)
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[交流]
【求助/交流】TSS法制備感受態(tài)的試劑選擇 已有4人參與
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| 小弟最近想用TSS法制備感受態(tài),請(qǐng)問(wèn)TSS法中的PEG,DMSO,Mgcl2和MgSO4都必須用進(jìn)口優(yōu)質(zhì)試劑嗎? 用國(guó)產(chǎn)的效果如何? 多謝! |
木蟲之王 (文壇精英)
我愛(ài)打老虎

木蟲 (正式寫手)
金蟲 (正式寫手)
銀蟲 (初入文壇)
銀蟲 (初入文壇)
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1. Afresh overnight culture ofbacteria is diluted 1:100 into prewarmed LB broth and the cells are incubated at 370C with shaking (225 rpm) to an OD600 of 0.3-0.4. 2. An equal volume of ice-cold 2x TSS is added and the cell suspension is mixed gently. [TSS is LB broth with 10% PEG (molecular weight 3350 or 8000), 5% DMSO, and 20-50 mM Mg2+ (MgSO4 or MgCl2) at a final pH of 6.5.] 3a. For long-term storage, cells are frozen immediately in a dry ice/ethanol bath and stored at -70°C. 3b. For transformation, a 0.1-ml aliquot of cells is pipetted into a cold polypropylene tube containing 1 ,ul (100 pg) of plasmid DNA, and the cell/DNA suspension is mixed gently. (When frozen cells are used, cells are thawed slowly on ice and used immediately.) 4. The cell/DNA mixture is incubated for 5-60 min at 4°C. 5. A 0.9-ml aliquot of TSS (or LB broth) plus 20 mM glucose is added, and the cells are incubated at 37°C with shaking (225 rpm) for 1 hr to allow expression of the antibiotic-resistance gene. 6. Transformants are selected by standard methods. |
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