1 st Strand Synthesis in Reverse Transcription

1. Random primer 5’ mRNA AAAAAAAA 3’ 3’ N6 N6 N6 N6 N6 1st strand cDNA Oligo(dT) primer AAAAAAAA 3’ mRNA PCR 5’ TTT TTTTT 5’ 3’ 1st strand cDNA Sequence specific primer mRNA AAAAAAAA 3’ 5’ 5’ 3’ 1st strand cDNA 1st Strand Synthesis in Reverse Transcription 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 MgCl2, 50mM DTT. • M-MLV RT is inactivated by heating at 70°C for 10 minutes.

3. RT Reaction Mix RNA samples: 1 2 3 4 5 WT, PDI2A-1, PDI2A-2, (-)No RT, Negative control (H2O) Group 1, 4 WT, GFP2SC, GFP5er, (-)No RT, Negative control (H2O) Group 2, 3 Reverse Transcription1X6X 1) RNase-free H2O ? μl ? μl 2) RT buffer (5X) 4.0 μl 24.0 3) RNase-free Dnase 0.5 μl 3.0 4) dNTPs (4mM each) 1.25 μl 7.5 5) Oligo(dT) primer (10 mM) 2.0 μl 12 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)

4. PCR Reaction Mix RT templates from RNA and control samples: 1 2 3 4 5 6 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 Reaction mix:1 X7 X RT template 5.0 µl --- ddH2O 13.1 µl 91.7 10x Immo buffer, 2.5 µl 17.5 dNTPs (25 mM), 0.2 µl 1.4 MgCl2 (25mM) 2.0 µl 14 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 µl 20 X7=140

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. Immolase is inactive at room temperature; it needs heat activation at 95C . Annealing temp : 56C 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

7. WT Pdi2A Pdi9A GFP WT-1 WT-2 2A-1 2A-2 1 2 1 2 3 7 2SC 5er MW - - At 1 2 3 1 2 3 1 2 3 1 2 3 MW PDI2 primers RNA Samples and RT-PCR -RNA DNase + + / + + - + + - + + - + + - RT + + / + - + + - - + - - + - - GFP2sc GFP5er - P+ 1 2 3 1 2 3 MW 1.2% gel with formaldehyde; Run at 60Volt GFP primers DNase / / + + - + + - RT / / + - - + - -