Haynes Lab:Notebook/Engineering PC-TFs/2013/10/25

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October 25, 2013

Sigma-Aldrich Mini-prep Kit

  1. Harvest cells: Pellet 1–5 ml of an overnight recombinant E. coli culture by centrifugation. 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.
  2. 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
  3. 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.
  4. 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.
  5. 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.
  6. 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.
  7. 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.
  8. 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.
  9. 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.

Measure concentration of each fragment:

  • Load Gen 5 2.0 software. Go to the Task Manager-Take 3 Application
  • Go to Nucleic Acid Quantification.
  • Pipette 2 µL of each DNA sequence into Take 3 plate.
  • Place plate in EPOCH plate reader.
Part Concentration
KAH126 281.975 ng/μL

Diagnostic Digest

Gel Electrophoresis

  • Orient top notch of gel electrophoresis apparatus. Place gel tray into larger tray.
  • Fill gel flask with up to 60 ml of TA buffer.
  • Create 1% gel by putting .6 grams of agarose into flask.
  • Microwave agarose solution for 40 seconds
  • Using hot gloves, grab flask out of microwave and swirl flask vigorously. Place back in microwave for 40 more seconds.
  • When flask is taken out of microwave, make sure that the agarose is completed dissolved.
  • Set up wells on gel electrophoresis apparatus. The mold for the first digest is B-10. Use the thick "teeth" and slip in the notches.
  • Add EtBr (ethidium bromide) to the agarose gel. There should be 1 microliter of EtBr per 10ml of agarose gel. Therefore, 6 µL.
  • Pour gel into tray.
  • Wash the agarose gel flask.
  • Run gel for 45 min at 100 V. Check gel under UV light.
Reagent Volume Diagnostic digest of KAH126
30 μL/lane, 1% agarose; Ladder
DNA (plasmid) 20.0 μL
XbaI 1.0 μL
PstI 1.0 μL
Green 10x FastDigest buffer 3.0 μL
dH2O 5 μL
  30 μL --> 37°C/ ~10 min.