RPM® 12G Protocol
Revision No. 2079-000-5G04W



Maximum prep capacity: 15 mg



Growth Media3 L LB*1 L CIRCLEGROW**
Inoculation Source6 ml Culture***2 ml Culture
Growth at 37°C14-16 hours1 to 3 days
Yield (High copy vectors)****10 - 30 mg****10 - 30 mg****
Yield (Low copy vectors)0.6 - 3 mg0.6 - 3 mg
*LB Broth (see pg 216)
**CIRCLEGROW is a super-rich bacterial growth media (see pg 212)
***Overnight culture
****High copy vectors = pUC, pTZ, pGEM, pBS, etc. Typical yield with insert is 1-3 mg.
Low copy vectors = pBR 322, cosmids, etc. Typical yield with insert is 0.1-0.5 mg.
Note: High copy vectors can become low copy with certain inserts. The notation "high" copy can only be used as a rough guide when estimating plasmid yield from a given culture volume or cell pellet weight. Yields from plasmids containing inserts that differ by a single base can vary in yield by as much as 100-fold.



    Modified Alkaline lysis; RNA degradation; Denaturation and Precipitation of proteins, cell debris and chromosomal DNA.
  1. Pellet culture at 6000 x g for 10 min (e.g., 6000 rpm in GSA rotor/4000 rpm in HG4L or TYJS 5.2rotor) and discard media.
  2. Resuspend cells in 40 ml distilled H2O by vigorous shaking and transfer to a 250 or 1000 ml centrifuge bottle. Add 4 ml Cell Wash Concentrate, swirl to mix and incubate 5 minutes at room temperature. Spin at 6000 x g for 10 minutes (i.e., 6000 rpm in GSA rotor) and decant supernate. Cell Wash is important in reducing endotoxin levels in the final purified DNA.
  3. Add 54 ml dH2O and 6 ml Pre-Lysis Concentrate. Mix by vigorously shaking until the cells are completely resuspended (This is essential for efficient cell lysis and maximal DNA yield).
  4. Prepare 60 ml Alkaline Lysis Solution in a separate container.


    5. H2OAlkaline Lysis IAlkaline Lysis II*
    46 ml8 ml6 ml


    * Before using Alkaline Lysis Concentrate II, dissolve precipitated SDS by warming, if necessary.

    Add to the cells and gently invert 15 times. Optional: incubate < 5 minutes at room temperature.

  5. Add 60 ml of ice cold Neutralizing Solution and shake 3-5 times until a uniform white precipitate forms. Incubate 10-30 minutes on ice and spin at 6,000 x g for 5 minutes at 4°C . Collect the supernate in a clean centrifuge bottle by decanting through a funnel lined with kit-supplied Sieve Material to filter out floating debris. (Sieve Material is reusable. Rinse well under running tap and blot dry after each use.)
  6. Add 500 µl RNase Mixx; invert to mix, and incubate 30 minutes at 55°C.
  • "GENECLEAN" purification of plasmid DNA
  • Add the content of one Binding Salt bottle and dissolve salt to completion by swirling several times. (Binding Salt contains guanidine thiocyanate; wear gloves and eye protection when handling.) Add the contents of one GLASSMILK 12G bottle; invert gently several times to ensure a uniform mixture. Incubate for 5 min at room temperature with occasional mixing (or continuous rocking) for efficient DNA binding to GLASSMILK 12G.
  • Spin at 2000 rpm for 1-2 minutes and discard the supernatant Add 20 ml of Wash Solution 12G (add ethanol before first use). Resuspend the GLASSMILK/DNA complex by gently stirring with a pipet, followed by gentle swirling. Transfer to Filter Unit 12 G attached to Bottle A and placed in Bottle Holder.


    • For faster vacuum filtration, remove the cotton plug from the side arm of Filter Unit 12G. The use of the Filter Unit 4G is an important part of this procedure and contributes significantly to the purity of the DNA which is especially important for in vitro and in vivo transfections. It also allows elution into a relatively small volume so that precipitation can be done in a single tube. Filtration time varies from 1-10 min depending on the vacuum source. A vacuum source (pump or house line) that provides 20-30 inches of Hg is optimal (>30 can damage membrane). During vacuum filtration use the Bottle Holder to stabilize the filter.




  • Connect to a vacuum source and filter the GLASSMILK/DNA solution until the GLASSMILK surface is exposed. Keep the vacuum on and add 10 ml of user-supplied 1:1 acetone/ethanol solution (5 ml acetone + 5 ml ethanol). When matrix surface is exposed, disconnect the vacuum and remove the Filter Unit 12G. Discard the filtrate in Bottle A which contains residual contaminants trapped by the GLASSMILK/DNA complex and removed during the washes.
  • Uncap the DNA Trap Tube; insert in Bottle B, and attach Filter containing the settled GLASSMILK-bound plasmid DNA. With the vacuum off, add 30 ml of 37°C autoclaved H2O; resuspend the GLASSMILK/DNA complex: dislodge the packed GLASSMILK 12G with the tip of a 10 ml pipet around the periphery of the filter, being careful not to break membrane. Gently loosen to form loose chunks and keep mixing into a slurry by stirring and swirling.
  • Apply vacuum to elute DNA solution into Trap Tube. When solution has filtered through, add 4 ml H2O and continue with the vacuum until the matrix surface is dry. Turn off the vacuum source; disassemble the filter unit; and discard the filter containing used matrix.
  • Precipitate the DNA
    1. Add 1.35 ml of 5 M NaCl and aliquot to two 50 ml centrifuge tubes. Add 12.5 ml of room temperature isopropanol per tube and invert to mix. Spin at 15,000 x g for 15 minutes at 4°C (11,000 rpm HB-4 or SS-34 rotor) and decant supernate. (Isopropanol pellets are transparent and, as a result, more difficult to see than the salt-containing ethanol pellets. To easily locate the pellet, mark the tube prior to centrifugation when using a fixed angle rotor).
    2. Add 5 ml of 70-80 % ethanol and swirl the tube to wash the pellet. It is not necessary to resuspend the pellet in the wash. Spin at 15,000 x g for 5 to 10 min at 4°C and carefully decant supernatant, keeping a close watch on the pellet which is easily dislodged at this step. Pulse spin (e.g. ~3000 rpm for <1 min in a tabletop centrifuge) and remove the last drop of ethanol with a pipet. Air dry 5 to 10 minutes at room temperature and dissolve the DNA in Isopropanol (pellets are transparent and, as a result, more difficult to see than the salt-containing ethanol pellets. To easily locate the pellet, mark the tube prior to centrifugation when using a fixed angle rotor).



    • Note: The use of a swinging bucket rotor is prefered and will ensure that the DNA pellet is collected at the bottom of the tube and not partially on the wall of the tube as with a fixed angle rotor. Isopropanol pellets are often somewhat transparent and, as a result, more difficult to visualize than salt-containing ethanol pellets. To easily locate the pellet when using a fixed angle rotor, mark the tube prior to centrifugation.


  • Air dry 5-10 minutes at room temperature and redissolve the DNA in 2 to 4 ml of autoclaved H2O/TE. (Caution: Solutions that have not been autoclaved and subsequently treated with sterile technique can contain active nucleases that will degrade plasmid DNA with time in storage). Store DNA at 4-20°C. DNA is ready for use without further manipulation.



    • Pause Points:

    Cell pellets can be frozen for extended periods (at least 6 months) before processing. Thaw at 37°C for a few minutes and continue with step 1 b.

    Isopropanol pellets in step 1 f. can be stored overnight in the refrigerator.



    If DNA is dissolved in H2O, add 5 µl TE to the cuvette containing the DNA sample in H2O when measuring the OD260/280. DNA purified by the RPM 12G is free of proteins, metabolites, RNA and genomic DNA, and contains low levels of endotoxin for efficient transfection studies.


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