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Protocol for Purification of Construct DNA for Microinjection

  1. From Endo-free maxiprep prepared plasmid DNA, digest overnight to destroy vector.
  2. Run digest on 1.2% low melting (genetic analysis grade) agarose gel in 1X TAE Buffer with 0.2% Ethidium Bromide
  3. Check progress of gel running with very low UV exposure
  4. Excise bands from gel and digest agarose with gelase enzyme (Epicentre) according to product insert “Fast Protocol.” (Stop following protocol after 1hr agarose digestion).
  5. Following agarose digestion, perform phenol extraction (1:1) (NOT phenol: chloroform)
  6. Remove aqueous layer and add 2 volumes isopropanol and 1/10th volume 3M NaOAc, pH 5.6; mix.
  7. Spin at 15,000 x g for 30 min-1 hr at 4°C
  8. Wash with 70% Ethanol and spin again for 10 min at 15,000 x g
  9. Air dry, and resuspend pellet in TE or H2O, pH 8.
  10. Run sample on 1.2% low melting (genetic analysis grade) agarose gel in 1X TAE buffer with 0.2% Ethidium Bromide
  11. Repeat steps 3-9.
  12. Measure sample concentration.
  13. After resuspension, run sample through Qiaquick Spin PCR purification kit (following product insert).
          Note: Do not overload column (Column capacity is 10 g)
  14. Elute in TE, H2O pH 8, or Buffer EB (10mM Tris-Cl).
  15. Measure/Confirm final sample concentration.


In the end, you should have performed two (2) gel purifications and one (1) column purification.