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.
