Alternatively, you may use the Qiagen QIAEX II gel extraction kit for fragments >50 bps.
Activation of DEAE membrane:
- cut DEAE membrane into 3-4" strips slightly thicker than the thickness of an average gel slab (they can be cut to desired size later)
- soak the strips 10 min. in 10 mM EDTA (pH 7.6)
- soak the strips 5 min. in 0.5 M NaOH
- rinse 6 times rapidly in distilled H2O
Strips can be stored several weeks in distilled H2O, RT or 4 C. Do NOT allow strips to dry.
Extraction of DNA band from gel:
Samples must be run on the gel and stained with EtBr for UV inspection. It is a good idea to run samples to be extracted with empty lanes on either side--this reduces the chance of contamination by unwanted DNA. Verify location of bands of interest and good separation of all bands before proceeding.
1. cut a slit (razor blades work well) slightly longer than the size of the band to be extracted immediately ahead of the band.
2. cut DEAE membrane to the size of the band to be extracted
3. with a blunt forceps, gently separate the gel at the slit and insert DEAE membrane completely (all the way to the bottom of the gel slab)
4. re-apply voltage (~120 V) and periodically monitor band progression with hand-held UV lamp
5. DNA will fluoresce on the membrane--this allows verification that the DNA is on the membrane
To ensure extraction of only the band of interest, perform steps 1 through 5, cutting the slit immediately behind (rather than ahead of) the band of interest. This traps the unwanted DNA. Then proceed per usual with these same steps as written.
6. remove the membrane from the gel and rinse it (allow to sit) in low salt buffer in a petri dish for ~3 min.
7. cut the strip into small pieces, ~2x2 mm, and place (not smash) them at the bottom of a 1.5 ml eppendorf tube; remove any in low salt buffer by pipet
8. add just enough high salt buffer to cover all membrane pieces. Elute 20 min. 65 C
9. transfer high salt buffer to a new tube and repeat step 8.
10. combine high salt buffers and phenol:chloroform extract
11. concentrate/desalt by microcon or EtOH precipitation (depending on size of DNA).
Buffer recipes:
Low salt buffer
0.15 M NaCl
0.1 mM EDTA
20 mM Tris
high salt buffer
1.0 M NaCl
0.1 mM EDTA
20 mM Tris
These buffers should be autoclaved.