PCR problem, no amplicon, tried everything I could think of.. - (Jan/23/2008 )
I've been trying to amplify the glucomannan synthase gene from P. radiata cDNA via PCR for a while now, but I don't seem to be able to get the correct amplicon. This is the first time I attempted to amplify from cDNA. The same reaction for amplification from plasmid DNA seem to work fine. Please help!
Things I have tried:
adding DMSO
increasing MgCl2
re-purify cDNA + oligo dT primer removal
total RNA was extracted from xylem tissue of the pine tree via CTAB, and using stratagene RT kit to make the cDNA.
My current PCR conditions are as follows:
Ultra pfu 10x Buffer - 3.7ul
forward primer (10uM) - 2ul
reverse primer (10uM) - 2ul
dNTPs (2.5mM) - 2.3ul
MgCl2 (10mM) - 2.5ul
ddH2O - 11.5ul
Ultra pfu polymerase - 1ul
Total volume - 25ul
Thermo cycler setting:
1. 95C for 5min
2. 95C for 30sec
3. 55C for 45sec
4. 72C for 60sec
5. cycle to step 2 for 32x
6. 72C for 10min
7. 4C for ever
I have also tried gradient PCR with annealing temp from 45C to 65C, and still didn't get the correct amplicon, just lots of side products (same when I increased the DMSO or MgCl2 too much).
The primers used are heterologous, from loblolly pine (very closely related specied, and glucomannan synthase genes should be identical)
Any help would be most appreciated! Thanks!
Things I have tried:
adding DMSO
increasing MgCl2
re-purify cDNA + oligo dT primer removal
total RNA was extracted from xylem tissue of the pine tree via CTAB, and using stratagene RT kit to make the cDNA.
My current PCR conditions are as follows:
Ultra pfu 10x Buffer - 3.7ul
forward primer (10uM) - 2ul
reverse primer (10uM) - 2ul
dNTPs (2.5mM) - 2.3ul
MgCl2 (10mM) - 2.5ul
ddH2O - 11.5ul
Ultra pfu polymerase - 1ul
Total volume - 25ul
Thermo cycler setting:
1. 95C for 5min
2. 95C for 30sec
3. 55C for 45sec
4. 72C for 60sec
5. cycle to step 2 for 32x
6. 72C for 10min
7. 4C for ever
I have also tried gradient PCR with annealing temp from 45C to 65C, and still didn't get the correct amplicon, just lots of side products (same when I increased the DMSO or MgCl2 too much).
The primers used are heterologous, from loblolly pine (very closely related specied, and glucomannan synthase genes should be identical)
Any help would be most appreciated! Thanks!
So, your PCR works with plasmid but not cDNA, have you checked your cDNA quality? ie, can you amplify other genes from it. Also, how much cDNA are you putting on your reaction, according to your recipe there's no volume left for it.
My bad My bad.. 
2ul of total cDNA (~100ng) is loaded into each PCR reaction
and its 9.5ul of ddH2O added in each PCR reaction.
If I am to check the quality of the cDNA to eliminate it as a variable, what primer set would you recommend to target which HKG in the cDNA?
Thank you very much!
I think Pfu amplification can be a little tricky in general. But your reaction mixture seems to be causing your problems.
> Pfu needs MgSO4 not MgCl2. The buffer that comes with the polymerase doesn't have MgSO4??
> Also your adding too much buffer. It should be diluted to 1X concentration i.e. 2.5 ul in a 25 ul reaction.
> Your primer concentrations are also high - 800 nM final conc. Maybe you need to optimise this?
Hope this helps! 
> Pfu needs MgSO4 not MgCl2. The buffer that comes with the polymerase doesn't have MgSO4??
> Also your adding too much buffer. It should be diluted to 1X concentration i.e. 2.5 ul in a 25 ul reaction.
> Your primer concentrations are also high - 800 nM final conc. Maybe you need to optimise this?
Hope this helps!
Thank you very much for the reply,
I did a Taguchi array to optimize the reaction condition, with a positive control as a similar gene but easier to amplify. and indeed the optimal conditions are:
Ultra pfu 10x Buffer - 2.5ul
forward primer (10uM) - 1ul
reverse primer (10uM) - 1ul
dNTPs (2.5mM) - 1ul
Betaine (5M) - 5ul
ddH2O - 12.8ul
cDNA - 1.5ul
Ultra pfu polymerase - 1ul
Thermo cycler setting:
1. 95C for 5min
2. 95C for 30sec
3. 54C for 45sec
4. 72C for 2min
5. cycle to step 2 for 32x
6. 72C for 10min
7. 4C for ever
This reaction condition successfully amplified the positive control, but when I changed the primer to target my gene of interest, it still failed.
Interesting though, if I use "Advantage" polymerase I was able to amplify the gene some times. but when I sequence the amplicons, due to the low fidelity, the sequence have 4~5 errors.
Any suggestions as to how I can amplify the target gene using the high fidelity DNA polymerase?
Thanks!
Redesign the primer. You could have tried three primers in the time you have fooled around with this one. The problem is almost certainly in the primer design. I use primer3 and check for primer hairpins and primer dimers with the IDT web site tool. The optimizations will not help if the primers are bad.
Agree with Phage.
Plus, i noticed in your first post that the primer you use are designed for another species of tree. You mentionned that the genes should be identical. So you should make sure that they are, and that the primers you use are designed for the right gene.
Plus, are you sure your gene is expressed? If not, you may well have some difficulties amplifiying it
Also, what is the lenght of the cDNA you are trying to amplify? If it's bigger than 1kb, your extension time may not be long enough (1 min per kb, usually).