Transgenic and Genome Manipulation Facility

  • Purification of Transgenic DNA for Microinjection

    QIAquick Gel Extraction Kit Protocol for Injection DNA Purification

    1. Digest 20 μg of plasmid DNA as necessary to cut apart the fragment and the DNA.
    2. Run on a 1% agarose gel until bands are well separated. Check quickly with a long wave UV light, keeping exposure to a minimum so as not to damage the DNA.
    3. Use a sterile scalpel to excise the desired band from the gel. Place gel slice in a 1.5 ml eppendorf tube.
    4. Weigh the gel slice. Place in a larger tube if necessary so that 3 volumes of Buffer QG can be added per 1 volume of gel (100 mg=100 μl). Ex. Add 300ul buffer per 100 mg gel
    5. Gently mix until gel slice has dissolved completely. The buffer should still be yellow. If the fragment size is <500 bp or >4000 bp add 1 volume of isopropyl alcohol and mix gently.
    6. Divide the mixture equally into two QIAquick columns (700 μl is the maximum load per spin). Spin for 1 minute at 13,000 rpm. Discard the flow-through and continue until all of the buffer mix has been applied to the columns.
    7. Wash 2X with 500 μl of buffer QG, discarding flow-through after each spin.
    8. Add 750 μl of Buffer PE to the columns and let sit for 5 minutes. Spin and discard flow-through.
    9. Wash twice more with 750 μl of Buffer PE, this time spinning immediately after adding to the columns.
    10. Discard all flow-through and spin for 5 minutes at 13,000 rpm to remove any remaining wash buffer.
    11. Place one of the columns in a fresh 1.5 ml eppendorf tube. Add 40 μl of filtered "Injection Buffer" directly over the center of the column.
    12. Let stand for 1 minute and then spin for 2 minutes at 13,000 rpm.
    13. Collect the flow-through and add it to the second column. Let stand 1 minute and spin as before.
    14. The purified injection DNA can then be quantified by spectrophotometer and gel analysis. Dilute as needed with filtered "Injection Buffer".
    15. Before each injection session, spin the DNA for 5 minutes at 14,000 rpm to pull down any particles that may block the injection needle.

     

    Injection Buffer

    • 500.0 μl 1.0M Tris-Cl pH 7.0
    • 10.0 μl 0.5M EDTA
    • 49.5 ml embryo-certified water
    • Autoclave and store at room temperature
    • Filter desired amount with a 0.45 μm Millex-HV syringe-driven filter unit before use.