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| PCR - Polymerase Chain Reaction Forum PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory. |
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#1
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| Can anyone help or advise me. I am a Summer Student and have been trying t amplify cDNA produced from RNA unsuccessfully for a number of weeks now. I performed the RT-PCR First Strand Synthesis using Fermatas Revert Aid Firrst Strand cDNA Synthesis Kit and after 10 attempts got a band using the following: 12ul MyTaq 1ul 18s Forward Primer 1ul 18s Reverse Primer 1ul concentrated cDNA 9.5ul Nuclease Free H2O This was to check there was actually cDNA produced. Primers were designed from either side of the "missing segment" of around 45 nucleotides. To amplify the gene in question (POR Gene) I have tried several things. Initially I used: 16.25ul Nuclease Free H20 5ul Buffer 0.5ul dNTPs 1ul Fwd Primer 1ul Rev Primer 1ul Template 0.25ul Enzyme (Phusion Hotstart II) PCR conditions used were 1 cycle: 98oC for 30 seconds 39 cycles of: 98oC 10 seconds 54oC 30 seconds 72oC 30 seconds 1 cycle: 72oC 5 minutes I have now tried altering the following: Changing to Velocity Enzyme Seeded PCR Halving the enzyme amount Reducing extension time Changing annealing temp from 54oC to 50oC and also 45oC Adding more Template Increasing annealing time to 60 seconds All of the above has produced no bands at all, or smears, or the PCR product still in the wells after gel was ran. I am really stumped now and would appreciate some advice. I hope I have included everything I need in this post. Many Thanks in advance. |
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#2
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| dear ff_fairy if you get best amplify I think you have to use mgcl2 with different concentration in PCR reaction for example : 5ul Buffer 2-8 ul mgcl2 (25 mm) (1mm -4mm ) 1ul dNTPs 1ul Fwd Primer 1ul Rev Primer 10ul cDNA 0.5ul Enzyme |
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#3
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| dear ff_fairy PCR conditions 1 cycle 95 oC ------- 60 seconds 40 cycles of: 95oC --------- 30 seconds 50-60oC ----- 30 seconds gradiant temperature 72oC ----------30 seconds 1 cycle 72oC --------- 5 minutes |
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| bands , pcr , product |
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