DNA precipitation

Hello everyone!

Regarding concentrating DNA, using salt and alcohol, what would be the

optimum combination of salt type (LiCl, NaAcetate, NaCl, potassium

acetate, etc) and alcohol type (isopropanol, ethanol 100%, 70%, etc.)?

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Hi! I did this twice several years ago, and it was successful so I won't be much help troubleshooting - but here's my protocol:

• Combine: o 0.1 volumes of 3M sodium acetate, pH=5.2 (e.g. 10µL) o 3 volumes of ice cold 100% ethanol (e.g. 300µL) o Vortex to mix • Precipitate at -20˚C for 1 hour or overnight (I did over the weekend) • Centrifuge at full speed, 4˚C for 30 minutes. • Wash pellet twice with 0.5 mL ice cold 75% Ethanol, spinning at 4˚C for 30 minutes each time o To wash, tip supernatant out, then gently tap drops down. • Air dry the pellet and resuspend in an appropriate volume of nuclease free water o This step can take hours, I left the open tube on top of my lab bench overnight.

I would just add to what David said: 1. The incubation time at - 20 C could be adjusted to get better results depending on the fragments of DNA that you are isolating. My supervisor used to say that most of the precipitation happens in one hour ~95 %, after that the increment by time is quite small. 2. To ressuspend the DNA I added miliq water and then did vigorous up-and-downs x20 and then incubated for 30 min at 37 C with agitation (with thermomixer). Since I began to do step 2 I was able to get better results in qPCR for chromatin immunoprecipitation experiments

Side note: I have found papers showing that cold precipitation is the same as room temperature. And also (but I did not find in papers) doing one hour at - 80 C would be similar to overnight - 20 C

Good luck.

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