基因工程原理实验指导 RNase-free DNase I1μl(1u/μ) 10×DNase I buffer1ul DEPC-treated ddH2O5 ul 4.Incubate at37℃forl5min,then70℃forl0min Reverse Transcriptase Reaction 1.Add 1 m1500 ug/ml oligo(dT)15 primer,mix the contents of the tube by gently vortexing and collect the reaction by brief centrifugation. 2.Heat the mixture at 65 C dry bath for 10 minutes,then place at room temperature for 10 minutes.Add the following contents: 5 x first strand buffer 4 ul 0.1MDTT2μl 10 m MdNTP1μl 3.Mix the contents of the tube by gently vortexing and collect the reaction by brief centrifugation.Place the tube at 37 C water bath for 2 min. 4.Add U/m I M-MLV Reverse Transcriptase.Mix gently and incubate at 37 C for I h.The total volume should now be 20 ul 5.Inactive the reaction by heating at 70 C for 15 min,then add 20ul ddH 2 O and the first strand of cDNA can be used as a template to amplification in PCR. 6.To remove the RNA complementary to the cDNA,add 1 ul(2 units)of RNase H and incubate at 37C for 20min PCR Amplification 1.Prepare the mixture containing the following on ice: cDNA first strand 1ul TaKaRa Ex Tag (5u/IuD05ul l0 x Ex Taq buffer5μl dNTPmixture(2mMW4μl Primer F(10 mM)2ul Primer R(10mM)2μl ddH2035.5μ1 2.Commence PCR program 94℃60℃72℃4℃Cycles Step 13 min 1 Step 2 Imin I min I min 30-40 Step35 min I Step 4 forever 基因工程原理实验指导 20 RNase-free DNase I １ µ l ( １ u / µ l) 10 × DNase I buffer 1 µ l DEPC-treated ddH 2 O 5 µ l 4. Incubate at 37 ℃ for 15 min, then 70 ℃ for 10 min. Reverse Transcriptase Reaction 1. Add 1 m l 500 µ g / ml oligo (dT) 15 primer, mix the contents of the tube by gently vortexing and collect the reaction by brief centrifugation. 2. Heat the mixture at 65 ℃ dry bath for 10 minutes, then place at room temperature for 10 minutes. Add the following contents: 5 × first strand buffer 4 µ l 0.1 M DTT 2 µ l 10 m M dNTP 1 µ l 3. Mix the contents of the tube by gently vortexing and collect the reaction by brief centrifugation. Place the tube at 37 ℃ water bath for 2 min. 4. Add 2 µ l 200 U / m l M-MLV Reverse Transcriptase. Mix gently and incubate at 37 ℃ for 1 h. The total volume should now be 20 µ l. 5. Inactive the reaction by heating at 70 ℃ for 15 min, then add 20 µ l ddH 2 O and the first strand of cDNA can be used as a template to amplification in PCR. 6. To remove the RNA complementary to the cDNA, add 1 µ l (2 units) of RNase H and incubate at 37 ℃ for 20 min. PCR Amplification 1. Prepare the mixture containing the following on ice: cDNA first strand 1 µ l TaKaRa Ex Taq (5 u / 1 µ l) 0.5 µ l 10 × Ex Taq buffer 5 µ l dNTP mixture (2 mM) 4 µ l Primer F (10 mM) 2 µ l Primer R (10 mM) 2 µ l ddH 2 O 35.5 µ l 2. Commence PCR program 94 ℃ 60 ℃ 72 ℃ 4 ℃ Cycles Step 1 3 min 1 Step 2 1min 1 min 1 min 30-40 Step 3 5 min 1 Step 4 forever