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1 st Strand Synthesis in Reverse Transcription AAAAAAAA 3’ N6 TTT TTTTT 5’ 5’ 3’ Random primer Oligo(dT) primer Sequence specific primer 1 st strand cDNA.

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Presentation on theme: "1 st Strand Synthesis in Reverse Transcription AAAAAAAA 3’ N6 TTT TTTTT 5’ 5’ 3’ Random primer Oligo(dT) primer Sequence specific primer 1 st strand cDNA."— Presentation transcript:

1 1 st Strand Synthesis in Reverse Transcription AAAAAAAA 3’ N6 TTT TTTTT 5’ 5’ 3’ Random primer Oligo(dT) primer Sequence specific primer 1 st strand cDNA mRNA 1 st strand cDNA mRNA 1 st strand cDNA mRNA 5’ PCR RT employs avian myeloblastosis virus (AMV) or Moloney murine leukemia virus (M-MLV) reverse transcriptases for first strand cDNA synthesis.

2 Promega Inc. Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) an RNA-dependent DNA polymerase used in cDNA synthesis with long messenger RNA templates (>5kb). a product of the pol gene of M-MLV and is a single subunit of 71kDa. its RNase H activity is weaker than Avian Myeloblastosis Virus (AMV) reverse transcriptase. 5X Reaction Buffer: 250mM Tris-HCl (pH 8.3 at 25°C), 375mM KCl, 15mM MgCl 2, 50mM DTT. M-MLV RT is inactivated by heating at 70°C for 10 minutes.

3 RT Reaction Mix Reverse Transcription 1X6X 1) RNase-free H2O ? μl? μl 2) RT buffer (5X) 4.0 μl24.0 3) RNase-free Dnase 0.5 μl3.0 4) dNTPs (4mM each) 1.25 μl7.5 5) Oligo(dT) primer (10 mM) 2.0 μl12 6) RNA (2ug) ? μl? μl Total volume 19 μl 7) briefly spin (10 sec), and incubate at 37ºC for 30 min for DNase to work, 8) briefly spin and transfer to 70ºC for 5 min (why ?), 9) add 1 μl of Reverse Transcriptase (M-MLV) to the tube (- total 20 μl), 10) mix and briefly spin. Incubate at 42ºC for 1 hour, 11) briefly spin and leave in 95ºC for 5 min (why ?). 12) add 30 μl of ddH2O to a final volume 50 μl. (Store at -20ºC if not to PCR) RNA samples: WT, PDI2A-1, PDI2A-2, (-)No RT, Negative control (H2O) Group 1, 4 WT, GFP2SC, GFP5er, (-)No RT, Negative control (H2O) Group 2, 3

4 PCR Reaction Mix Reaction mix: 1 X7 X RT template 5.0 µl --- ddH2O13.1 µl x Immo buffer, 2.5 µl17.5 dNTPs (25 mM), 0.2 µl 1.4 MgCl2(25mM) 2.0 µl14 Primer 1 (100ng/µl) 1.0 µl 7 Primer 2 (100ng/µl) 1.0 µl 7 Immolase DNA Polymerase 0.2 µl 1.4 (5 U/ul) Total volume 25 µl20 X7=140 RT templates from RNA and control samples: WT, PDI2A-1, PDI2A-2, (-) No RT, (-) control (H2O), (+) control Group 1,4 WT, GFP2SC, GFP5er, (-) No RT, (-) control (H2O), (+) control Group 2,3 1 ul plasmid DNA 4 ul H2O

5 Immolase from Bioline Features and applications: Heat-activated (- preincubation at 95ºC for 7 minutes ) Extremely high specificity Elimination of non-specific reaction products Polymerises regions of DNA such as secondary structures or microsatellites, which are difficult to extend with other polymerases. Storage Conditions: IMMOLASE DNA Polymerase can be stored at -20°C, in a constant temperature freezer for 9 months. IMMOLASE will remain stable if stored as specified. 10x Immo Buffer: 10x ImmoBuffer 160mM (NH4)2SO4, 1M Tris-HCl pH 8.3, 0.1% Tween-20) Storage and dilution buffer: 20mM Tris-HCl, pH 7.5, 100mM NaCl, 0.1mM EDTA, 2mM DTT, 50% Glycerol, and 0.1% Tween-20

6 PCR and Sequencing PCR conditions: 95  C for 7 min 35 cycles 95  C for 30 seconds Tm – 5  C, 30 seconds 72  C, extension 1 min/kb 72  C, 7 min 4  C, ~~ Purify PCR products and sequencing Immolase is inactive at room temperature; it needs heat activation at 95  C. Annealing temp : 56  C

7 RNA Samples and RT-PCR SC 5er WTPdi2APdi9AGFP 1.2% gel with formaldehyde; Run at 60Volt MW - - At MW PDI2 primers WT-1WT-22A-12A-2 DNase + + / RT + + / P MW GFP primers DNase / / RT / / GFP2scGFP5er -RNA


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