Help with PCR and Gel Electrophoresis. Not getting any bands - (Sep/07/2012 )
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.
How were your primers designed? Anything special about the template (high GC, low GC?, structure)?
I already have 2 sequences, one is the 5' end of the gene, and the other the 3' end. There is however a gap of around 45 nucleotides in the middle which is what I am trying to amplify. The primers were designed from these sequences either side of the gap, are 20 nucleotides long and not particularly GC rich.
Those look like perfectly reasonable primers. How do you know the length of this gap? Are you able to amplify the known regions of your gene from the same template sample?
Perhaps the gap is really an intron, and you have been amplifying genomic DNA rather than cDNA.
Did a Blast search and the compared the 2 sequences as from same family, using ClustalW2 and got an idea of the length of the gap.
And, can you amplify the portions you have sequence for from your cDNA template?
You could amplify genomic DNA with the two primers you have. If you get a short result, then you will cover the gap. If you get a long result, you will know there is an intron. What is the end goal? Are you trying to make the gene? If so, then you can probably substitute one of the homologous sequences you have apparently found for the 9 aa that are missing.
The sequences I already have were given to us along with several others from another research dept and when we did a BLAST search of them all in the database, the two we have are the ones that came up as the POR Gene but there is a small sequence missing in between them.
So, I would recommend that you try to amplify the sequences you already know are present. This will check that your cDNA (or perhaps genomic DNA) really is present and of sufficient quality. I would also try dilutions of your template.
I have already tried dilutions of the template, but will try the sequences I already know, thank you