Haynes Lab:Notebook/Engineering PC-TFs/2013/10/24

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October 24, 2013

Harvest cells: Pellet 1–5 ml of an overnight recombinant E. coli culture by centrifugation. The optimal volume of culture to use depends upon the plasmid and culture density. For best yields, follow the instructions in the note below. Transfer the appropriate volume of the recombinant E.coli culture to a microcentrifuge tube and pellet cells at 12,000 x g for 1 minute. Discard the supernatant. Note: For best results with recombinant E. coli grown in LB (Luria Broth), use 1–3 ml of culture for high copy plasmids or 1–5 ml of culture for low copy plasmids.

  1. Resuspend: cells Important Reminder: Verify that appropriate volume RNase A Solution was added to the Resuspension Solution. Completely resuspend the bacterial pellet with 200 µl of the Resuspension Solution. Vortex or pipette up and down to thoroughly resuspend the cells until homogeneous. Incomplete resuspension will result in poor recovery.Another rapid way to resuspend the cell pellets is to scrape the bottoms of the microcentrifuge tubes back and forth 5 times across the surface of a polypropylene microcentrifuge tube storage rack with 5 x16 holes.^3
  2. Lyse cells: Lyse the resuspended cells by adding 200 µl of the Lysis Solution. Immediately mix the contents by gentle inversion (6–8 times) until the mixture becomes clear and viscous.Do not vortex. Harsh mixing will shear genomic DNA, resulting in chromosomal DNA contamination in the final recovered plasmid DNA. Do not allow the lysis reaction to exceed 5 minutes. Prolonged alkaline lysis may permanently denature supercoiled plasmid DNA that may render it unsuitable for most downstream applications.
  3. Neutralize: Precipitate the cell debris by adding 350 µl of the Neutralization/Binding Solution. Gently invert the tube 4–6 times. Pellet the cell debris by centrifuging at 12,000 x g or maximum speed for 10 minutes. Cell debris, proteins, lipids, SDS, and chromosomal DNA should fall out of solution as a cloudy, viscous precipitate. If the supernatant contains a large amount of floating particulates after centrifugation, recentrifuge the supernatant before proceeding to step 6.
  4. Prepare: Column Insert a GenElute Miniprep Binding Column into a provided microcentrifuge tube, if not already assembled. Add 500 µl of the Column Preparation Solution to each miniprep column and centrifuge at 12,000 x g for 30 seconds to 1 minute. Discard the flow-through liquid. Note: The Column Preparation Solution maximizes binding of DNA to the membrane resulting in more consistent yields.
  5. Load cleared lysate Transfer the cleared lysate from step 3 to the column prepared in step 4 and centrifuge at 12,000 x g for 30 seconds to 1 minute. Discard the flow-through liquid.
  6. Optional wash (use only with EndA+ strains). Add 500 µl of the Optional Wash Solution to the column. Centrifuge at 12,000 x g for 30 seconds to 1 minute. Discard the flow-through liquid.
  7. Wash column: Important Reminder: Verify that ethanol has been added to the bottle of Wash Solution 2. Add 750 µl of the diluted Wash Solution to the column. Centrifuge at 12,000 x g for 30 seconds to 1 minute. The column wash step removes residual salt and other contaminants introduced during the column load. Discard the flow-through liquid and centrifuge again at maximum speed for 1 to 2 minutes without any additional Wash Solution to remove excess ethanol.
  8. Elute DNA Transfer the column to a fresh collection tube. Add 100 µl of Elution Solution or molecular biology reagent water to the column. For DNA sequencing and other enzymatic

applications, use water or 5 mM Tris-HCl, pH 8.0, as an eluant. Centrifuge at 12,000 x g for 1 minute. The DNA is now present in the eluate and is ready for immediate use or storage at –20 °C. Note: If a more concentrated plasmid DNA preparation is required, the elution volume may be reduced to a minimum of 50 µl.