質粒DNA提取時質粒為何會丟失？

aw852755 2013-10-13 16:59:22 628 瀏覽

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天天聽音樂go 2013-10-14 00:00:00

中穩定存在，經多次轉接后有可能造成質粒丟失。因此不要頻繁轉接，每次接種時應接種單菌落。另外，檢查篩選用

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翻譯protocol（有關質粒DNA提取，堿裂解法）？: 請哪位高人幫忙找道這篇的中文：PreparationofPlasmidDNAbyAlkalineLysiswithSDS:Minipreparation（題）或者幫忙翻譯一下，要求不要用電腦直接翻譯，總之通順準確即可。急需截止25號。... 請哪位高人幫忙找道這篇的中文：Preparation of Plasmid DNA by Alkaline Lysis with SDS: Minipreparation（題）或者幫忙翻譯一下，要求不要用電腦直接翻譯，總之通順準確即可。急需截止25號。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. 展開

質粒dna提取時為什么加入sds和naoh后會出現粘稠:

質粒DNA提取過程都需無菌操作嗎:

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質粒DNA提取時質粒為何會丟失？

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