[問題] DNA 電泳

看板Biotech (生命科學)作者hchingling (星光)時間19年前 (2006/05/16 20:13)推噓1(1推 0噓 0→)

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我的電泳圖除了有目標band 沒有其他雜band 可是背景卻是一整條像是拖尾的東西就連負控制都是如此原以為是萃dna時沒有將蛋白質處理好於是在phenol那個步驟多萃幾次（直到中間介面沒有白色樣出現）在最後的步驟再加入1λRNase於37℃下作用10分鐘實在是想不出還有什麼方法可以改善了請各位幫忙了看看是不是我有什麼是沒注意到地因為沒有相簿可以放如果要看圖可能要給我你的msn或是mail 麻煩各位了以下是我的protocol 1. Inoculate a 5 ml liquid culture of bacterial strain of interest in appropriate media. Grow until culture is saturated. 2. Centrifuge = 1.5 ml of the culture to collect pellet (2 min in microfuge).Remove supernatant. 3. Resuspend pellet in 567 ul TE buffer. Add 30 ul of 10% SDS and 3 ul of 20 mg/ml proteinase K (final [ ]'s of 0.5% and 100 ug/ml, respectively).Mix thoroughly and incubate 1 hr at 37℃. 4. Add 100 ul of 5 M NaCl and mix thoroughly. 5. Add 80 ul of CTAB/NaCl solution. Mix thoroughly and incubate 10 min at 65℃ . 6. Add an approximately equal volume (0.7 - 0.8 ml) chloroform/ iso-amyl alcohol (24:1), mix thoroughly, and centrifuge for 5 min in microfuge. (see white interface) 7. Remove aqueous, viscous supernatant to a fresh microfuge tube, leaving the interface behind. Add an equal volume of phenol/chloroform/iso-amyl alcohol (25:24:1), mix thoroughly, and centrifuge for 5 min in microfuge. 8. Transfer the supernatant to a fresh tube. Add 0.6 vol isopropanol to precipitate the nucleic acids (enough salt present).Rock tube back and forth until stringy white DNA ppt becomes clearly visible. At this point it may be possible to transfer the pellet to a fresh tube containing 70% EtOH with a micropipette. Alternatively, the ppt can be pelleted by briefly spinning at RT (room temperature). 9. Wash the DNA with 70% EtOH to remove residual CTAB and respin 5 min at RT to repellet it. Carefully remove the supernatant and briefly dry the pellet under vacuum. 10. Redissolve the pellet in 100 ul TE buffer. This may take up to 1 hr. Typically, 15 ul is sufficient for digestion and running out on an agarose gel in order to give a good signal during a Southern hybridization. -- ※ 發信站: 批踢踢實業坊(ptt.cc) ◆ From: 140.117.91.148