rt-pcr（rt - pcr）I. Experimental apparatus and materials:1, transfer guns: 1ml, 200 L, 20 L, 10 L, 2 L2, suction head: 1ml, 200 L, 20 L3, homogenate tube: 5ml4, suction head table: put 1ml suction head one, put 20 l suction head one5, EP tubes: 1.5ml, 0.2ml, 100 L6, reagent bottle: 2 60ml Brown reagent bottle (wide mouth, with cover)1 125ml white reagent bottles (with absolute ethanol)7, 50ml, 250ml, 500ml: a8, capacity bottle: 250ml, 500ml, 1000ml9, test tube rack: 5ml, 1.5ml, 20 L10, salt water bottles: 250ml, 500ml each 2 spare, one with absolute ethanol, another with DEPC water11, aluminum lunch box: 412, plastic small lunch box: 113, large porcelain cylinder: 214, tin paper: a roll15 rolls of paper: 2 rolls16, triangle flask: with a cap, slightly largerTwo, the processing and preparation of experimental equipment1, plastic products: (including gun head, EP tube, homogenate tube, etc.)The DEPC of water from the flask into the ceramic cylinder, the plastic products by soaking them, which requires a small tip Straw DEPC into the water, and then dried overnight, high pressure, spare, before the experiment will first put the gun suction head, and a high pressure (EP tube)2, glass products: acid soaking overnight, washed clean, dry spare foil Mongolia (DEPC blister) (wash after the first bubble 1 per thousand DEPC overnight, and then dried)3, homogenizer: (including scissors, tweezers) first wash, and then high pressure (do not need to bubble DEPC)Three. Reagent preparation:1, DEPC water: suck 1ml, placed in 1000ml double steamed water, with 1 per thousand DEPC water, placed in the 1000ml capacitybottle, static 4 hours standby.2, 75% ethanol: with anhydrous ethanol DEPC water, and then put -20 degrees preservation (where DEPC water should be high pressure)3, isopropyl alcohol: put brown bottle4, chloroform: put in brown bottle5 agaroseFour. Preparation of several buffers:1 Electrophoresis buffer:Tris 54GBoric acid 27.5g0.5M, EDTA, 20ml? PH8.0Distilled water 1000ml5 x TBE (Zhu Cunye)The 5 x TBE is diluted 10 times to 0.5TBE, which can be used in electrophoresis (i.e., the concentration of the working fluid), such as 50ml storage solution, 450ml water, 500ml working buffer2, the sample buffer:0.25% bromo phenol blue0.25% xylene green FF30% glycerin6 * buffer solution, stored at 4 DEG CFive. Preparation of agarose gel:1 and 1%:1.0g 100ml agarose electrophoresis buffer, microwave oven fire 30 seconds to boiling, adding2.5 L agar 10mg/ml ethidium bromide cooled to 60 DEG C melting, mixing, will warm into the gel film has been set a good comb, placing 30-45min at room temperature after the electrophoresis.2 and 1.5%:Ditto, change the amount of agarose to 1.5gSix, the purchase of Rt-PCR materials:(working man. Tel. 2236106.)Taq enzyme (containing MgCl2, Buffer) 200u 70DNTP: 1Oligo (dT) 15: 1 OD 40 PromegaM-MLV: 1, 250, Promega (Buffer)RNasin: 1, 110---20 DEG CDEPC 5g 110Trizol 100ml/1 bottles Invitrogen Life technologies - 4 degrees centigradeMarker: 10 g 0.2 g/ml 150 Kuwait(1543., 994., 697., 515., 377.231)Seven. Primer synthesisJustice: 5 '-CACGATGGAGGGGCCGGACTCATC-3' Antisense: 5 '-TAAAGACCTCTATGCCAACACAGT-3'2, par-4:Justice: 5 '-GGGACCTCGGAACTCAAC-3'Antisense: 5 '-TGTATCTGCCTGGGACTG-3'3. Calculation of annealing temperature2 (A, T) 4 (G, C)The average number of positive and negative averages fluctuates again and again (+ 4 DEG C, or + 5 DEG C)4. The primers are 5 OD each, and each OD bottle is packed separately5 primer dilution:The amount of water added to DEPC is (L)= nmol / OD * on the tube marked OD * 100Is a primer solution for 10p mol / L concentrationEight and PCR electrophoresisThe first 1-10 L PCR products, and has little on paper by LAN, repeatedly playing suction after mixing by electrophoresis, the general current of 10mA, power 100V, electrophoresis 30 minutes, observed under ultraviolet light, satisfactory results for scanning and printing.Nine, several points of attention:1, the key step of reverse transcription is immediate ice water bath2, Rt, first open PCR preheat 30 minutes.3, RNA pumping ahead, open the centrifuge pre cooling.Total RNA was extracted by TRIzolCells 1 * 107Tissue 100mgHerePlus 1mlTRIzolThe cells were blown to the liquid with a 1ml sampler and clarified without cell massHereHomogenate (thoroughly, then transferred to the EP tube)(tissue homogenate >100mg 1ml/ EP tube each time)HereMix it upside down 10 times, room temperature for 5 minutesHereChlorination of 1/5 volume (0.2ml) (must press the total volume of 1/5)HereMix it upside down 10 times, room temperature for 5 minutesHere4 DEG C, centrifuged 12000g, 15 minutesHereTransfer to the upper aqueous phase (about 400 l) in another 1.5mlEP tubeHereAdd an equal volume of isopropyl alcohol (about 400 l) and mix it for 10 minutes at room temperatureHere4 DEG C, centrifuged 12000g, 10 minutesHereSupernatantHere75% ethanol (DEPC water) 1ml with iceHereCentrifuge at 7500g for 4 minutes for 5 minutesHereDiscard the supernatant and let the air dry for 5-10 minutes (not completely dry)HereSoluble in DEPC water to 20 l (10 L-20 L)(water soluble at <10 for 55-60 minutes.)Note:1, RNA if used for nucleic acid transfer, should be dissolved in the sample buffer, or DEPC dissolved2, the cell tissue plus TRIzol homogenate can be placed at least -60 months (or more than a year)3, RNA in 75% ethanol, 2-8 DEG C can be stored for at least one week, -20 DEG C can be stored for at least one yearTwo step RT-PCR(step 1: reverse transcription)Concentration of reagentRNA 23 Mu L (11.5 L)Oligo (dT) 150.05 mu g/ Mu L, 4 Mu L (2 L) 0.005 mu g/ Mu L (diluted 10 times)HereMixing, centrifugation, 70 5minHereImmediately ice bath, slightly centrifugalHereConcentration of reagentM-MLV Buffer 5 * 8 L (4 L) 1 *DNTP 10mM 2 mu L (1 L) 0.5mMRNasin 40U/ Mu L 1 mu L (0.5 L) 20 muM-MLV 200U/ Mu L 2 mu L (1 L) 200UThe total volume is 40 L (20 L)HereMixing, centrifugation, 42 60minHere95 10min (destroy MLV)Here4 degrees preservationTwo step RT-PCR(second step: PCR reaction)The total volume is 20 l (50 L)Concentration of reagentTaq Buffer 10 * 2 L (5 l_) 1 *MgCl2 25mM 1.2 Mu L (3 L) 1.5mMDNTP 10mM 0.2 Mu L (0.5 L) <200uMUpstream primers 10pmol/, Mu L, 0.3 Mu L (1 L) 10pmol Downstream primers 10pmol/, Mu L, 0.3 Mu L (1 L) 10pmol CDNA template X () (1-10 L)DEPC water 20 l-4.3 l-XTaq enzyme 2.5U/ Mu L, 0.3 Mu L (1 L) 2.5U/ Mu L HereMixHere97 degrees, 5 minutesHereInstant ice bathHereMixHerePre denaturation at 95, 594 DEG C, 30 second degenerationAnnealing at 40 X72 degrees, 30 seconds extension72 point, 7 point, end extension28-36 cycle, 4 degree heat preservationThree electrophoresis:Add 1-10 Mu L PCR product bromine Finland (1-2.5 L) mix, add sample, electrophoresis.Note: Taq enzymes, M-MLV and Rnasin are stored at -20 DEG C and placed on ice during operation. DNTP do not freeze again and again.。