DNA concentration-IPA

What follows is a detailed protocol for λDNA concentration. I usually concentrate the entire product of a λDNA handle ligation, and I recommend using at least 5 mL. For standard small-volume ethanol precipitation, see Ethanol Precipitation.

Contents

1. Reagents
2. Protocol

1. Reagents

• 3M Sodium Acetate
• Isopropanol

2. Protocol

2.1 Day 1: Estimated Time- 1.5 hour

1. Add 1:10 (10%) volume:volume 3M Sodium Acetate to λDNA handle ligation product.
2. Flash freeze IPA using liquid nitrogen for 1 minute. Place in ice bucket and use immediately.
3. Add 7:10 (70%) volume:volume cold IPA to DNA solution.
4. In a fixed-angle rotor, centrifuge using 15 mL tubes at 15,000 × g for 30 minutes at 4°C (NOTE: MARK THE AREA LIGHTLY WITH A SHARPIE).
5. Look for the pellet, it should appear as a small white dot on the side of your conical. Try to keep track of it during the rest of the protocol, it is easy to lose.
6. CAREFULLY decant supernatant, and wash the pellet 2x in 2 mL of 70% EtOH.
7. Centrifuge using 15 mL tubes at 15,000 × g for 15 minutes at 4°C.
8. CAREFULLY decant supernatant.
9. Air dry the pellet for about 30 sec - 1 min.
10. Dissolve the pellet in 50 uL TE.
11. Place in 37°C water bath 4h.
12. Nanodrop the concentrated DNA from the top, middle, and bottom of the tube.
13. If readings are not consistent, place in the 37°C water bath O/N.