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