A little late but here is an update on the work that Bri and I worked on last Tuesday. Picking up from the chelex extraction that Carson and Allison performed and the chelex Proteinase-K extraction that Anna performed, our goal for the night was to set up and hopefully run a nucleic acid precipitation of their extractions. While we can use the DNA extracted from the chelex protocol in downstream applications like PCR, the resin in the chelex prevents accurate absorbance-based DNA quantification. Our hypothesis is that the nucleic acid precipitation would help purify the DNA and allow us to use the donated spectrophotometer we have in lab currently as a quality control check.
This protocol works by relying on the fact that DNA is slightly polar. The slightly polar charge of DNA causes DNA to be less soluble in non-polar alcohol than they are in polar water. Additionally, the phosphate-sugar backbone of DNA, which is responsible for DNA’s slight negative charge, can be neutralized with the addition of salt making the DNA less hydrophilic and thus more able to precipitate.
The protocol we used was adapted from OpenWetWare
1. 95% isopropanol
2. 3M sodium acetate pH 5.2
3. PCR grade water
4. -20C Freezer
1. Weigh volume of Chelex-extracted DNA solution
2. Add 1/10th volume of 3M sodium acetate pH 5.2
3. Add 2.5 volumes of 100% ethanol
4. Place in -20 freezer overnight
5. The next day, microcentrifuge at 13,000 x g for 30mins to pellet DNA
6. Decant the supernatant
7. Air dry for 15-20mins
8. Resuspend the pellet in 50uL of PCR-quality water