碱裂解法提取质粒过程溶菌酶的作用

对牛弹琴冲淡尚 2012-10-22 17:22:08 349 浏览

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梅眉辣挤诱犊 2012-10-23 00:00:00

跟SDS一样，裂解细胞壁释放质粒用的。不过不加也没什么问题，NaOH与SDS足够将大肠杆菌细胞壁裂解开。

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goolring 2012-10-24 00:00:00

碱裂解法的裂解液中起主要裂解作用的是NaOH，NaOH可以将细胞膜裂解开，对于革兰氏阴性菌，如大肠杆菌，这类菌，细胞壁很薄，细胞膜裂解后，该菌体就会裂解，所以含有NaOH的裂解液裂解效果明显。但对于革兰氏阳性菌，如乳酸菌，这类菌细胞壁较厚，NaOH无法进行有效裂解，而溶菌酶主要通过破坏细胞壁中的N-乙酰胞壁酸和N-乙酰氨基葡糖之间的β-1，4糖苷键，使细胞壁不溶性黏多糖分解成可溶性糖肽，导致细胞壁破裂内容物逸出而使细菌溶解。当然，对于这种难裂解的细胞，加溶菌酶或煮沸都是一种有效的裂解方法。

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热门问答

碱裂解法提取质粒过程溶菌酶的作用:

碱裂解法提取质粒DNA时的SDS有什么作用:

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翻译protocol（有关质粒DNA提取，碱裂解法）？: 请哪位高人帮忙找道这篇的中文：PreparationofPlasmidDNAbyAlkalineLysiswithSDS:Minipreparation（题）或者帮忙翻译一下，要求不要用电脑直接翻译，总之通顺准确即可。急需截止25号。... 请哪位高人帮忙找道这篇的中文：Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation（题）或者帮忙翻译一下，要求不要用电脑直接翻译，总之通顺准确即可。急需截止25号。原文如下： 1. Inoculate 2 ml of rich medium (LB， YT， or Terrific Broth) containing the appropriate antibiotic with a single colony of transformed bacteria. Incubate the culture overnight at 37°C with vigorous shaking. 2. Pour 1.5 ml of the culture into a microfuge tube. Centrifuge at maximum speed for 30 seconds at 4°C in a microfuge. Store the unused portion of the original culture at 4°C. 3. Remove the medium by aspiration， leaving the bacterial pellet as dry as possible. 4. Resuspend the bacterial pellet in 100 μl of ice-cold Alkaline lysis solution I by vigorous vortexing. 5. Add 200 μl of freshly prepared Alkaline lysis solution II to each bacterial suspension. Close the tube tightly， and mix the contents by inverting the tube rapidly five times. Do not vortex! Store the tube on ice. 6. Add 150 μl of ice-cold Alkaline lysis solution III. Close the tube and disperse Alkaline lysis solution III through the viscous bacterial lysate by inverting the tube several times. Store the tube on ice for 3-5 minutes. 7. Centrifuge the bacterial lysate at maximum speed for 5 minutes at 4°C in a microfuge. Transfer the supernatant to a fresh tube. 8. (Optional) Add an equal volume of phenol:chloroform. Mix the organic and aqueous phases by vortexing and then centrifuge the emulsion at maximum speed for 2 minutes at 4°C in a microfuge. Transfer the aqueous upper layer to a fresh tube. 9. Precipitate nucleic acids from the supernatant by adding 2 volumes of ethanol at room temperature. Mix the solution by vortexing and then allow the mixture to stand for 2 minutes at room temperature. 10. Collect the precipitated nucleic acids by centrifugation at maximum speed for 5 minutes at 4°C in a microfuge. 11. Remove the supernatant by gentle aspiration as described in Step 3 above. Stand the tube in an inverted position on a paper towel to allow all of the fluid to drain away. Use a Kimwipe or disposable pipette tip to remove any drops of fluid adhering to the walls of the tube. 12. Add 1 ml of 70% ethanol to the pellet and invert the closed tube several times. Recover the DNA by centrifugation at maximum speed for 2 minutes at 4°C in a microfuge. 13. Remove all of the supernatant by gentle aspiration as described in Step 3.Take care with this step， as the pellet sometimes does not adhere tightly to the tube. 14. Remove any beads of ethanol that form on the sides of the tube. Store the open tube at room temperature until the ethanol has evaporated and no fluid is visible in the tube (5-10 minutes). 15. Dissolve the nucleic acids in 50 μl of TE (pH 8.0) containing 20 μg/ml DNase-free RNase A (pancreatic RNase). Vortex the solution gently for a few seconds. Store the DNA solution at -20°C. 展开

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