Plasmid Mini-Prep Protocol

  1. Inoculate 5ml of medium containing the appropriate antibiotic with a single bacterial colony (use tweezers to pick up a sterile toothpick, touch the colony with the tooth pick and drop it in the medium). Incubate overnight at 37o C with vigorous shaking to aerate.
  2. Prepare resuspension buffer (Buffer R) in 15ml orange falcon tube:

    Buffer R
    Item and Final Concentration Amount for 12 samples (1.5mL)
    50mM glucose 150_l 0.5M Glucose
    10mM EDTA 30_l 0.5M EDTA
    25mM Tris-Cl (pH 8) 37.5_l 1M Tris-Cl pH 8
    Water 1.28ml H2O (2 x 640_l)
    Note: If cells produce lysosyme then none has to be added, otherwise 4mg/ml should be introduced

  3. Fill an eppendorf to within a millimeter or two from the top with the overnight culture by simply pouring it, avoid contaminating the stock culture. Centrifuge for 1 minute at max speed. Store the remaining culture at 4o C.
  4. Remove the medium by aspiration. Make sure to get residual on inside of cap, and avoid directly touching the pellet. After finishing all samples, clean the aspirator by running 7% bleach through it. This helps avoid growth in the collection flask as well as the rubber tubing.
  5. Resuspend in 100_l Buffer R and place tubes on ice. Can resuspend by either vortexing 2 tubes together or manually using a pipette (which works a little faster). Note: Make sure that the pellet is completely resuspended and no clumps are left because once the lysis solution is added in the next step, resuspension is impossible due to the increased viscosity.
  6. Add 200_l lysis solution (Buffer P2 in QIAquick kit-0.2N NaOH, 1% SDS). Add to each tube individually or in small groups and invert rapidly 2-3X. Note: Don't vortex, this avoids shearing of chromosomal DNA. Make sure to mix reasonably quickly following addition of lysis solution, and check to make sure that lysis has occurred. This can be done by opening the cap. The solution should be stringy and viscous because of chromosomal DNA. A translucent string of ooze should connect the open cap and tube. Solution should turn somewhat clear following this step.
  7. Add 150_l, ice cold Potassium Acetate pH 4.8 (should be in fridge) to each tube individually and mix before adding to next. Vortex inverted at low RPM, reinvert to get any KAc that it stuck in the bottom of the eppendorf, and gently vortex inverted. The solution should look like egg drop soup. Store on ice for 5 minutes.
  8. Centrifuge for 5 minutes at RT, then pour the supernatant into a fresh eppendorf. No need to remove all SN with a pipette, just make sure that you have about 400 + _l.
  9. Add 400_l (1 volume) chloroform (fridge in orange tube) to each tube individually, and vortex immediately, i.e. before adding to the next sample. Then vortex all tubes again just prior to centrifugation. Centrifuge 2 minutes at RT. Note: The stock bottle has 2 phases, an upper aqueous and lower unaqueous, which is what we want. To obtain only lower phase push the plunger on the pipette to the bottom and then let is suck up a little air. Then put it down into the desired layer push out the puff of air and suck up the desired liquid.
  10. Remove 400_l the aqueous phase using a P1000 to a new eppendorf tube, this will be the final tube in which the DNA will be stored. Note: Remove 400_l only, forget about the rest and avoid the interface. Place tip on side of tube and slide it down with the decreasing liquid level. If any of the lower face is removed it will be apparent by the presence of two layers in the pipette tip. If two layers are apparent just put the lower and a little of the upper back into the tube.
  11. Add 800_l (2 volumes) EtOH (95% @ RT). Vortex and then centrifuge for 5 minutes. Note: Following vortexing the tubes should be left @ RT for two minutes but no longer, so to save time following vortexting place sample in the centrifuge before moving on to the next.
  12. Pour off 95% EtOH and place inverted on a paper towel. Add 1ml 70% EtOH and gently invert 2-3x. Note: Drip the 70% EtOH in on the side opposite the pellet to avoid dislodging it. If the pellet does come dislodged, re-centrifuge for 1min and proceed as above.
  13. Pour off the 70% EtOH, then give a brief spin to remove any residual EtOH off the sides of the tube and remove the remaining with a pipette being careful not to touch or dislodge the pellet.
  14. Dry pellets in Speed Vac, approx. 5 minutes. Can tell samples are dry when the vacuum gauge reads 50 or below.
  15. Resuspend in 20_l TE + RNAse, and perform restriction digest with appropriate enzyme to confirm presence and orientation of fragment.