RNA to VCU
From Protists
RNA to VCU
Contents |
Background
- salt (3M Sodium Acetate (NaOAc), ethanol and cold temperatures allow us to precipitate out RNA to ship it safely with minimal degradation and shearing
- this protocol is really the first step of RNA extraction, but it is all that is required of our lab to safely ship the RNA to VCU (as per their instructions to us)
Before You Get Started
- Check to be sure that we have 3M Sodium Acetate (NaOAc) or make new stock
- 100mL orange-capped media bottle
- ~500uL 3M NaOAc pre-aliquoted 1.5mL RNase-free tubes in the -20C (Check Lab Stocks Box)
- Check to be sure that we have 100% molecular grade ethanol (EtOH; in the flammables cabinet: Sigma Aldrich E7023-500mL)
- Use barrier pipet tips and sterile (RNase-free) plasticware for this protocol
- Buy dry ice from CSS (just prior to when you will start protocol):
- bring a clean/Styrofoam container from our lab (preferably one that fits securely into a cardboard box) to CSS
- check the weight of the container itself on the scale at CSS and note the difference after you add the dry ice to tell the check out person
- charge it to the VCU grant account (ends in 313)
- remember the weight of the dry ice + the container to tell the CBIO personnel for shipping weight estimate
- keep lid on dry ice at all times when you are not putting your samples in!
- Get regular ice from 6th floor ice machine
- this needs to be finished & up to the CBIO office before 3pm & must allow one business day of travel (i.e. DO NOT ship on a Friday or before a federal holiday or the dry ice will melt and the RNA will be ruined in transit)
RNA Precipitation & Preparation for Shipment to VCU
- Thaw out RNA to ship from -80C on regular ice (~5min)
- Add 3M NaOAc to each individual RNA rinse tube you will be shipping, flick to mix and immediately return to ice
- need a final concentration of 0.3M NaOAc in each tube (1:10 dilution factor), so total volume divided by nine equals the amount of 3M NaOAc you should add to dilute it appropriately
- assume ~27uL of RNA in each rinse tube because of loss onto membrane during spin down and amount removed to nanodrop (27uL/9 = 3uL, therefore, 3uL of 3M NaOAc should be added to each rinse tube)
- Add 100% molecular grade EtOH to each individual RNA rinse tube you will be shipping, flick to mix and immediately return to ice
- need to add enough EtOH to be ~2.5 times the RNA volume
- assume 30uL of RNA (27uL RNA in RNase-free water + 3uL 3M NaOAc), so 30uL x 2.5 = 75uL, therefore, 75uL of EtOH should be added to each rinse tube)
- be sure labels on tubes are legible and parafilm caps well
- put tubes in a labeled plastic ziplock baggie (name, # tubes, OTUs included, date, lab phone #, etc.) and try to cover the baggie with dry ice as much as possible (use cold-protective gloves)
- tape up Styrofoam container with packing tape and put it in cardboard box (if available) and tape that up as well
- bring container up to CBIO office before 3pm and let them know:
- ship package Fedex Next Day AM
- ~ weight of package (dry ice + container)
- address to ship to: Dr. Andrey V. Matveyev, Dept. of Microbiology & Immunology, Virginia Commonwealth University, 1101 East Marshall St., Rm 5036 Sanger Hall, Richmond, VA 23298, Phone: (804) 628-3068
- charge it to the Farmer VCU account (ending in 313)
- your name, x38111 for questions if they have any
- email Andrey to let him know shipment details (avmatveyev@vcu.edu) and can also CC the email to Mark Farmer (farmer@cb.uga.edu) and Greg Buck (gabuck@vcu.edu) to keep them abreast of project progress from our end
- OTUs sent
- rinses sent/OUT
- ~total [RNA]/rinse; this can be estimated by multiplying the value in the ng/uL column by the ~# of uL in the tube (~27uL): ex. 12.71ng/uL x 27uL = 340ng of RNA in this rinse tube; 405.94ng/uL x 27uL = 11,000ng or 11ug of RNA in this rinse tube
- attach ND-1000 .txt file for his information
- note any error or notes about readings or samples during Nanodrop of RNA in the email text
Farmer Lab Links
