3M Sodium Acetate (NaOAc)

(Difference between revisions)

Revision as of 15:07, 9 March 2009

This is a salt that we use to neutralize the charges on the sugar phosphate backbone of RNA to make the RNA far less hydrophilic and therefore much less soluble in water (specifically, the Na+ ions neutralize the negative charge on the PO3- groups on the nucleic acids)
This allows us to precipitate out RNA (in combination with ethanol and cold temperatures) to allow us to ship it safely with minimal degradation and shearing

Check to be sure that we are out of 3M NaOAc before you make new stock
- 100mL orange-capped media bottle
- ~500uL 3M NaOAc pre-aliquoted 1.5mL RNase-free tubes in the -20C (Check Lab Stocks or Misc. Boxes)
Rinse out bottle that previously had NaOAc in it (above PCRleft bench station) with HPLC or RNase-free molecular grade water and then do a final rinse with Molecular Grade Ethanol (~100% in the Flammables Cabinet) and allow to dry on paper towels
Autoclave prepared bottle for 30min on Gravity cycle
Be sure that we have DEPC-treated RNase-free water (in 4C walk in or -20C) or make more DEPC Water with the DEPC in the 4C drawer (be careful of built up pressure/explosion and only work with DEPC in the hood with appropriate safety protection)
Use barrier pipet tips and sterile (RNase-free) plasticware for this protocol

10.205g NaOAc (use the big bottle of 2.5kg NaOAc Trihydrate from Darley in the chemical cabinet; J.T. Baker 3460-05)
add ~15mL of DEPC Water (RNase-free) to weigh boat to get all crystals out and into the autoclaved bottle prepared above
shake like hell until all crystals are dissolved
add ~5.8mL glacial acetic acid (in bin under hood where acids are stored) and check on pH paper (4.7-6.8 range in live side culture materials cabinet with other pH paper) â�� Note: this might stay at ~5.3pH for ~2min (~accurate) and then after that it changes back to dark blue, which indicates >6.8pH (this is not accurate)
add ~8-9 NaOH pellets to the acidic NaOAc and shake like hell until they are completely dissolved
Check standards of 4.0 and 7.0 buffers with the pH meter to be sure close enough, or re-standardize the machine with those two buffers (manual next to machine has instructions)
Put ~4mL of the well-mixed NaOAc into a 15mL orange-capped centrifuge tube (just enough to cover probe sensor)
Measure pH accurately using pH meter (ok if between 5.2-5.4pH, otherwise adjust with glacial acetic acid or NaOH pellets as needed)
Autoclave on liquid cycle for 15-20min
Allow to cool and aliquot into 500uL/1.5mL RNase-free tubes with labels and put in the Lab Stocks box in the -20C

these are notes so you can estimate how much NaOH pellets are required for the pH change you require
5.24 was the final pH for the frozen aliquots and current contents of the media bottle of 3M NaOAc above the PCRleft bench station

followed directions as above (up to 5.8mL glacial acetic acid from PBJBrownâ��s glass bottle)
1. pH = 4.8
2. added 1 NaOH pellet and rechecked pH = 4.93
3. added 4 NaOH pellets and rechecked pH = 5.11
4. added 2 NaOH pellets and rechecked pH = 5.24 (ok at this)