ff_fairy

I will try the touchdown PCR thank you for your suggestions. I have not adjusted the salt concentration at all no. I will try the touchdown PCR thank you for your suggestions. I have not adjusted the salt concentration at all no.

Can anyone help or advise me. I am a Student and have been trying to amplify cDNA produced from RNA unsuccessfully for a number of weeks now in order to complete a gene sequence where I have a small gap for around 40 nucleotides. 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 then 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.