Download RNA Extraction Protocol

RNA Extraction Protocol
How to make Solution D
Add 200 micro-liters of mercaptoethanol to 30 ml of Solution D
Date bottle, it will be good for several weeks
SOLN D  Into 100ml ADD
47g --- Guanidium Hyocyanate
0.735G --- Na Citrate
0.5g --- Sarco Sye
pH = 7.0
This experiment is done at room temperature
1. 400 ul of Solution D in a tube
a. Add to this the cells that have been solubilized in H2O
b. Add 10 ul of glycogen form -20°C fridge.  200µg
c. Add 40 ul of NaAcetate (mix by shaking with hand)  3M at pH 5
d. Add 400 ul of Phenol (in 4°C fridge) - press the pipette as you are going through the
top layer
e. Vortex
2. Sheer DNA with a needle 5-6 times  Clean needle with ETOH for reuse
a. Add 160µl of 49:1 IsoamylETOH : Chlorophorm to soln
b. Vortex for 30 sec on Max
c. Place Isopropanol on ice from 4°C fridge
d. Lable new tubes
e. Centrifuge old tubes in Small centrifuge on Max Speed
3. Take the supernatant and place it in New tubes
a. Add 200 ul of isopropanol  Mix by hand
b. Place @ -20°C for 1 hour (Can keep them here for three days)
c. Centrifuge tubes @ Max for 15 min
4. Aspirate with suction tube but watch out for pellet
a. Add 70% ETOH (100ul) to resuspend
b. Centrifuge for 15 min @ Max Speed
5. Take off ETOH
a. Speed Vac Dry (No heater, Dry Rate Low)
b. Resuspend in Q'PCR H2O or DPC H2O 30 ul. (ON ICE HERE)
RNA Concentration Measurement:
1. Dilute 5ul in 900 DH2O ( Dilution factor is 180X)
2. Cubes for spec - blue squares facing out
3. Set reference - Cube with the H2O
4. Press ABS for the Absolute Value and Ration for the 260/280 Value
RNA Concentration Calculation:
1. Take Abs value @ 260nm X 9000 = ug/L
2. Divide this value by 1000 = ug/ul
3. Need 300 ng/gene Amplification in RT PCR
4. Can just multiply absorption by 9 to get the final value.