For isolation of DNA less than 50 kb in length
1. Add 60 µl of binding buffer to 25 µl of DNA sample to be purified in a microcentrifuge tube.
2. After resuspending glass using a plastic transfer pipette, add 15 µl of glass concentrate. Tap tube briefly to mix the contents.
3. Let stand at room temperature for 10 min. Invert the tube at one minute intervals.
4. Centrifuge at 7000 x g for 30 s in a microcentrifuge. Discard as much supernatant as possible using a transfer pipette.
5. Add 300 - 500 µl of wash buffer (300 works well). Using a non-shear pipette tip (supplied with kit), tease the glass pellet from the wall of the microcentrifuge tube. Gently pipette glass pellet up and down until pellet becomes focculent. Invert tube once. (Note: achieving a flocculent pellet is important for buffer exchange).
6. Centrifuge at 7000 x g for 30 s. Discard supernatant.
7. Repeat steps 5 & 6.
8. Add 300-500 µl (300 works well) of salt reduction buffer. Gently resuspend glass pellet until flocculent with the non-shear pipette tip as described above. Invert the tube once, and centrifuge at 7000 x g for 2 minutes. Discard supernatant.
9. Centrifuge glass pellet at 7000 x g for 30 s to condense pellet. Using a 20 µl pipette tip, carefully withdraw as much residual supernatant as possible and discard.
10.Add 70 µl of 1X TE or distilled, deionized water to glass pellet and resuspend by flicking the tube repeatedly. Incubate mixture at 50C for 5 min, occasionally flicking the tube. Centrifuge at 7000 x g for 30 s in a microcentrifuge and withdraw supernatant containing the isolated DNA to a fresh tube.
11. Quantify concentration prior to use.
P. O. Box 2012
Keene, NH 03431
My recommendation is that you compare the results of PCR experiments with uncleaned vs cleaned template and between undiluted and diluted templates. Optimization of conditions for each primer should follow these initial experiments.
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