分子生物學(xué)實驗experimentsofmolecularbiology-資料下載頁
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【導(dǎo)讀】《分子生物學(xué)實驗》課程主要從提取和分析遺傳物質(zhì)和基因為基礎(chǔ)，從DNA的分離、純化、克隆到目的基因的鑒定、表達(dá)等過程設(shè)計了一。分子生物學(xué)的相關(guān)理論知識，并較系統(tǒng)地學(xué)習(xí)和掌握分子生物學(xué)的基本實驗方法、技術(shù)和操作技能。通過學(xué)習(xí)使學(xué)生對所有實驗內(nèi)容有一個整體的了解；熟悉分子生物學(xué)實驗技術(shù)（包括DNA技術(shù)、RNA技術(shù)、蛋白質(zhì)技術(shù)、雜交技術(shù)等）的基本原理方法，掌握DNA提取純化,定量,DNA克隆,要求學(xué)生分子生物學(xué)實驗實驗室操作安全與污染物處理；學(xué)習(xí)儀器設(shè)備的使用。堿裂解法提取質(zhì)粒是根據(jù)共價閉合環(huán)狀質(zhì)粒DNA和線性染。色體DNA在拓?fù)鋵W(xué)上的差異來分離質(zhì)粒DNA。
【正文】 s. ? 1) Tissue is homogenized as rapidly as possible, at 4176。C, in solution D (500ul per 50mg tissue) with an eppendorf pestle homogenizer until a smooth, lysed, homogenous suspension is obtained. ? 2) Add 50ul 2M sodium acetate, and mix vigorously. ? 3) Add 500ul phenol and mix vigorously. ? 4) Add 100ul chloroform, mix vigorously and incubate on ice for 15 minutes. ? 5) Centrifuge mixture at 10,000g for 10 minutes in a microfuge at 4176。C. ? 6) Remove upper, aqueous phase to a clean, sterile, DEPCtreated eppendorf tube. After centrifugation, RNA is present in the aqueous phase while, due to protonation at the acidic pH used, genomic DNA is partitioned into the phenol phase. ? 7) Extract the upper aqueous layer with an equal volume phenol/chloroform and centrifuge as before. Repeat the extractions until no interface material is seen. ? 8) Precipitate the aqueous phase by the addition of an equal volume (500ul) of propan2ol. Incubate at 20176。C for 20 minutes. ? 9) Pellet RNA by centrifugation at maximum speed in a microfuge for 10 minutes. ? 10) Wash the RNA once in 70% ethanol and vacuum dry. ? 11) Redissolve in 200ul % SDS at 65176。C. ? 12) Extract with an equal volume (200ul) of phenol/chloroform as above. Repeat until no interface material is visible. ? 13) Precipitate pure RNA by the addition of 20ul 3M sodium acetate, 100mM acetate, pH and 500ul absolute ethanol. Incubate at 20176。C for 20 minutes. ? 14) Pellet RNA by centrifugation at maximum speed in a microfuge for 10 minutes. ? 15) Wash the RNA once in 70% ethanol and vacuum dry. ? 16) Dissolve RNA in appropriate buffer . DEPCtreated, sterile TE, pH 8 or % SDS if no enzymic manipulation of the RNA is needed. SDS is an inhibitor of ribonucleases. ? RNA quality can be assessed by electrophoresis under denaturing conditions using agarose/formaldehyde gels and the MOPS buffer system.
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