Oocyte isolation, maturation, enucleation and fractionation

1. Pick your embryos (~5 per tube) and place in an autoclaved 1.5 ml capped centrifuge tube
2. Wash embryos 1 ml of autoclaved DEPCed-PBS (containing 1x PBS; pH 7.4) -- take care not to lyze or damage embryos. (Wash embryos 3X if you are using RNAlater).
3. Add 1 ml of TRIzol reagent (located in the 4æC fridge next to Chi’s bench). Homogenize sample by pipetting up and down so sample is uniform and/or vortex). Note: cell remnants remain on the inside of the tips if immediately start homogenizing and you can’t get the remains off - perhaps vortex first.
4. Let sit at room temperature for 10 minutes. (Cells are being lysed at this stage).
5. Spin tubes in microfuge at 14 Krpmfor 10 minutes at 4æC.
6. Remove the supernatant (RNA) and place into a new autoclaved 1.5 ml tube. Add 200 µl of chloroform (located in the hood near Chi). Discard old tube (and pellet)
7. Vortex for 15 seconds and let sit at room temperature for 3 minutes.
8. Spin in microfuge at 12K for 15 minutes at 4æC.
9. Remove the top liquid layer (RNA; clear layer -- not the pink layer) and place into a new autoclaved tube. Use the hood to do this because of the chloroform.
10. Add 500 µl of isopropanol (wash) and invert to mix. Let sit for 10 minutes at room temperature.
11. Spin in microfuge at 12K for 10 minutes at 4æC. Note placement of tube in centrifuge, so you know where your pellet is -- the pellet contains the RNA!
12. Remove supernatant carefully with pipette leaving pellet -- pellet may be difficult (impossible) to see, do not despair!
13. Wash pellet with 1 ml RNAase-free 75% ethanol (made with DEPC water and absolute ETOH).
14. Recover RNA by centrifugation (9Krpm) for 5 minutes at 4æC).
15. Remove supernatant and air dry tube for 10 minutes (leave tube uncapped upside down at a slant; after 10 minutes look for residual dots of alcohol -- if see them air dry a bit longer).
16. Once dry, dissolve pellet in 30 µl of DEPC water.
17. Check RNA concentration : Add 100 µl of DEPC water and 1 µl of your isolated RNA to new tube.
    Check RNA concentration using spectrophotometer (make sure that dilution factor in the machine in the Lee lab is 100 (do blank first). Blanks should be with DEPC water using rna-free tips so as not to contaminate your DEPC water container.
18. Run Gel (look for ribosomal RNA bands).

DEPCed H2O

100 µl of Diethyl Pyrocarbonate (in fridge “Sauron”) + 100 ml of dH20) into a 100 ml glass bottle
stir for 30 min. and allow to sit overnight. in the 37 degree room

Autoclave to remove DEPC
Allow to cool (40 minutes - 1 hour) and then use as normal H2O
while making solutions

DEPCed PBS
90 ml of DEPCed water
+ 10 ml of (10x) PBS (big white jug near eyewash)
Autoclave. . Store at room temperature.

75% ETOH-DEPC
Use gloves.
7.5 ml of ETOH (ethanol)(stored in the yellow cabinet for flammable substances)
+ 2.5 ml of DEPC H20

1. Wash (if using RNA later). Note: you can proceed directly to homogenization and then store lysed homogenates at -20 C or -80 C.
2. Remove RNAlater (be careful not to expose embryos to air).
3. Wash embryos with DEPC-PBS.
4. Lyse. Add 0.6ml lysis to 5 oocytes/embryos Place 5 oocytes or embryos per tube in .6 ml of the lysis buffer. Can store homogenates @ -20 C or -80 C.
5. Add 20 µl of Proteinase K (flick, no vortex) (located in “Darth Vader” and labeled “Prok”)
   Digest for 1 hour at 37C or 55C (or up to 1 week at room temperature).
6. Add 700 µl (0.7 ml) of aqueous phenol (located in “Lux Luther”; Tamara may have in “Sauron”).
7. Centrifuge for 5 minutes at 14000 rpm at 4C. After, just remove the top layer.
8. Add 700 µl of phenol-chloroform (in fridge labeled “Sauron”-- lowest shelf-- the label is rubbed off). TAKE from the very bottom -- don’t take the upper chloroform layer. Centrifuge again 5 minutes at 14000 rpm at 4C. Just take top layer from tube.
9. Add 70 µl (1/10 volume) of 3M Na acetate (pH 5-55). If this is not available, can use 70 µl of 5 M ammonium acetate from the mMessage Machine Kit (any of the three versions of the kit).
10. Add .7 ml (700 µl) of isopropanol. Put in -20C fridge for one hour or preferably overnight
11. Thaw and then Centrifuge for 15 minutes for 14,000K at 4C.
12. Remove all supernatant from the pellet by aspiration. You should be able to see the pellet. Dry out.
13. Resuspend in 56 µl DEPC H2O + 24 µl of 10 M LiCl. If use the mMessage Machine kit, that kit is 7.5 M LiCl, so instead of 24 µl, use 34 µl (Final will be 3M LiCl).
14. Incubate -20C for 1 hour to collect precipitate.
15. Centrifuge for 15 minutes at 14000 rpm at 4 C
16. Remove supernatant (pellet will be clear). Save the supernatant if you are nervous or want the DNA. (You can precipitate the DNA with ethanol).
17. Rinse pellet with 70% (Ethanol + DEPC water). Pellet will turn white -- Dry.
18. Add 50 µl of DEPC water. Run on the gel using RNA procedure:
    1 % argose gel (RNase-free spray the plate before- watch out and be careful with plat wires at either end). No need to change the TAE).
    3 µl RNA ladder from Tamara (in “Agent Smith” fridge).
    2-3 µl of RNA + 2 µl of RNA running buffer from the mMessage Machine kits
    Heat final tubes in 70 C -90 C bath for 2-5 minutes to get the RNA to denature before putting in gel.
    Put directly on ice.
    Load gel
19. Check RNA concentration (as above)