RT-PCR

(RNA extraction with RNA-STAT) 1. Mix six embryos with 800 µl of RNA-Stat. Note. Amount of Total RNA are 3-4 µg/embryo and 300-400 ng/AC. 2. Add 200 µl of choloroform and recover the supernatant (100 µl, mix very well). 3. Centrifuge at maximum speed (e.g. 15k rpm) for 10 min at 4 ºC. 4. Recover the supernatant (usually 400 µl) and do a chloroform extraction. 5. Save 380 µl of supernatant and add 380 µl of isopropanol. 6. Mixed well and incubate in dryice until freeze or in -80 ºC for 30 min. Note. You can store sample in -20 ºC forever. 7. Centrifuge at maximum speed for 15 min at 4 ºC. 8. Wash the pellet with 70% EtOH and dissolve pellet into 12 µl of water. Note. Use 5 µl for the following Northern Blot reaction. (Loading Sample) Components 20 µl final Master Mix. RNA Water 5 x RT buffer d[N]6 (100 mM) dNTP (5 mM) DTT (0.1M) RNA guard BSA (1 mg/ml) MMLV RT enzyme 4 µl 6 µl 6 µl 3 µl 3 µl 3 µl 0.6 µl 3 µl 1.5 µl --- 300 µl 300 µl 150 µl 150 µl 150 µl --- µl 150 µl --- If you have 8 samples---- 1. Mix 24 x 8.5 µl of Master Mix, 0.6 x 8.5 µl of RNA guard, and 1.5 x 8.5 µl of MMLV RT enzyme very well (Called as Master Enzyme Mix). 2. Mix 4 µl of RNA solution and 26 µl of Master Enzyme Mix very well. 3. Incubate at 42 ºC for 1 h. 4. Add 30 µl of water. 5. Store at -20 ºC (Use as soon as possible. Do not keep for more than 3 months). (PCR) Components 20.4 µl final Master Mix. cDNA solution 10 x PCR buffer (with Mg) dNTP (5 mM) α-32P-dCTP FW-Rv-Mixed primers (each 20 mM) Water Taq polymerase 3 µl 2 µl 0.8 µl 0.1 µl 2 µl 12.2 µl 0.3 µl --- 480 µl 192 µl --- --- 2928 µl --- In the case that you have 8 samples and want to check 6 markers for each samples-------Please always refer to the letter color, above table, and right figure. (Set up the following under the cold condition) 0-1. Set up PCR machine and prepare six 8-connected PCR tubes. 0-2. Put 3 µl of cDNA soln into PCR tube and keep them in 4 ºC. 0-3. Put 2 x 8.5 µl of primers into 1.5 ml tubes (total six tubes). RADIO ACTIVE START from HERE!!! BE CAREFUL!!! 1. Mix 15 x 9 x 6 µl of PCR Master Mix, 0.3 x 9 x 6 µl of Taq polymerase, and 0.1 x 9 x 6 µl of α-32P-dCTP (called as PCR Master Enzyme Soln). 2. Mix 15.4 x 8.5 µl of PCR Master Enzyme Soln into 1.5 ml tubes with primers (see 0-3) very well (called as PCR Master Enzyme&Primer Soln). 3. Put 17.4 µl of PCR Master Enzyme&Primer Soln into each PCR tube (see 0-2). 4. Add one drop of mineral oil into each PCR tubes. 5. Close tubes and start PCR. 6. Run on a 5 % acrylamide gel after PCR. Components Volume Water 40% acrylamide 10x TBE APS TEMED 33 ml 5 ml 3.3 ml 500 µl 30 µl 5% acrylamide gel

Major markers: Stage 9 Wnt target Chordin Noggin Xnr3 Siamois Twin Pintallavis ADMP Nodal target Cerberus Xnr1 Xnr2 Xnr5 Xnr6 Sox17α Mix1 Milk Mixer BMP target Vent1 Vent2 Msx1 Msx2 ID BCNE center Chordin Noggin Xnr3 Siamois Twin Pintallavis ADMP Nieuwkoop center Cerberus Xnr1 Xnr2 Xnr5 Xnr6 Stage 13-14 Good for AC & VMZ assay Cement Gland XAG Anterior Neural Otx2 Six3 Pan-neural NCAM Sox2 Dorsal Mesodermal MyoD Myf5 Xbra Epidermal Cytokeratin Ventral Mesodermal Bmp4 Sizzled Crossveinless-2 Vent1 Vent2 Msx1 Msx2 Wnt8 Stage 18-22 Good for VMZ assay Ventral Sizzled β-Globin Paraxial PAPC Neural crest Slug Axial Shh Pintallavis Floor plate F-Spondin Dorsal α-Actin Cement Gland XAG Heart Nkx2.5 Stage 22-26 Good for AC assay Pan-neural NCAM N-Tubulin Anterior Neural Six3 Otx2 Pax6 RX2a Mid-Hind-brain boudary Engrailed Hindbrain Krox20 Posterior Neural Xlhbox6 (=HoxB9) Mesoderm α-Actin β-Globin Cement Gland XAG Loading Control: ODC: Always good loading control marker EF1α: Much stronger than ODC but not good before gastrula.