质粒DNA提取时质粒为何会丢失？

aw852755 2013-10-13 16:59:22 596 浏览

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天天听音乐go 2013-10-14 00:00:00

中稳定存在，经多次转接后有可能造成质粒丢失。因此不要频繁转接，每次接种时应接种单菌落。另外，检查筛选用

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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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