Miniprep/Qiagen kit protocol

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< Miniprep
Revision as of 04:52, 23 September 2009 by Vaishnavi Ananth (Talk | contribs)
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Solutions/reagents:

Equipment:

  • Centrifuge
  • Microfuge
  • Flasks of appropriate volumes
  • Sterile 1.5-ml microcentrifuge tubes

Steps:

  1. Measure out 1.5 ml of bacterial culture into a sterile 1.5-ml microcentrifuge tube.
    Centrifuge at maximum speed for 1 min at room temperature, gently aspirate out the supernatant and discard it.
    Add 250 µl of ice-cold Buffer P1.
    Resuspend the pellet by vortexing/by shaking vigorously.
    Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
  2. Add 250 µl of Buffer P2.
    Close the tube tightly and invert the tube 4 - 6 times times.
    Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear.
    NOTE: Proceed to the next step within 5 mins!
  3. Add 250 µl of Buffer N3.
    NOTE: Proceed to the next step within immediately!
  4. Close the tube tightly and invert the tube 4 - 6 times times.
    To avoid localized precipitation, mix the solution gently but thoroughly, immediately after addition of Buffer N3. The solution should become cloudy.
  5. Centrifuge at a speed of 13000 rpm for 10 mins at room temperature and aspirate out the top layer.
    Transfer top aqueous layer into QIAprep spin column.
    Discard bottom layer.
    A compact white pellet will form the bottom layer.
    Microfuge column for 30 - 60 secs.
    Discard the flow-through.
    Centrifuging for 60 seconds produces good results.
  6. (Optional)
    Add 0.5 ml of Buffer PB to column.
    Microfuge column for 30 - 60 secs.
    Discard the flow-through.
    This step is necessary to remove trace nuclease activity when using endA+ strains such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content. Host strains such as XL-1 Blue and DH5?™ do not require this additional wash step. Although they call this step optional, it does not really hurt your yield and you may think you are working with an endA- strain when in reality you are not. Again for this step, spinning for 60 seconds produces good results.

  7. Add 0.75 ml of Buffer PE to column.
    Microfuge column for 30 - 60 secs.
    Discard the flow-through.
    Centrifuging for 60 seconds produces good results.
  8. Microfuge column for 1 min.
    Discard the flow-through.
    This is to remove the residual wash bufer.
    IMPORTANT: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions. They are right about this.
  9. Transfer column into a clean 1.5 ml microcentrifuge tube.
    Add 50 µl of Buffer EB to column.
    Store at room temperature for 1 min.
    Microfuge for 1 min.
    Discard the column.
    The flow-through contains the DNA.
    If you are concerned about the concentration of the DNA, you can alternatively add 30 µl water to the center of the column, incubate at room temperature on the bench for 5 mins and then centrifuge for 1 min. This will increase the concentration of DNA in your final sample which can be useful in some cases. See notes below for why you should elute in water rather than the Buffer EB they recommend if you plan to sequence your sample. Even if you are not sequencing, it may be beneficial to elute in water. For instance, if you elute in buffer EB and you are using this DNA in a restriction digest, then the additional salts in your sample can affect the salt content of your digest. This may matter with some finicky enzymes.

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