Hi
Please, if the add of extra primer (add 100 picomol/microliter instead of 10 pm/microliter ) can affect the positive results?
I can see a couple of things that might affect PCR, lets have a look and see if we can sort this out.
10pM seems a bit low for a primer solution, giving you about 500fM in the final PCR. I would recommend increasing this to about 10µM, which would give you about 500nM in the final PCR. Primers are often the limiting factor in terms of PCR yield, as they are used up in every cycle and oce they run out the reaction cannot continue.
The extension duration looks ok, but you might want to increase it a bit, as the High-Fidelity polymerases tend to be a bit slower than Taq, which is usually quoted as 1000 bases/min, so maybe increase this to 5 min. Don't worry about degrading the template/polymerase, as 72°C should not affect them too much in a PCR mastermix.
Rather than increasing, I would probably decrease the amount of template you are adding. In your first experiment, you have about 40,000 copies of the genomic template DNA and in your second this increases to 94,000 genome copies. The risk here is that there is a large amount of other DNA present that can competatively bind to your primers (even though the binding is very weak because the base pairs don't match, there is alot of it) and reduce their chances of being used to prime your polymerase extension. I would reduce you input DNA amount to about 5,000 genome copies (approximately 4 ng/reaction, dilute 5µL of your stock DNA in 195µL of nuclease free water and use 5µL of this per PCR).
I am guessing that you are using 30 cycles as you want to reduce the risk of introducing mutations into the sequence so you can use it for ligations or something similar. Yes, this can be a problem, but it is more important to have a higher yield, so if the above steps didn't work, increase your number of cycles to 35. Every cycle that you run will give you approximately double the amount of PCR product, so even a modest increase in cycle number can improve yield dramatically.
If none of that improves your yield, try digesting your genomic DNA template with a restriction enzyme that does not cut inside your PCR product. If you get to this, let us know and I will help you pick one
The extension duration looks ok, but you might want to increase it a bit, as the High-Fidelity polymerases tend to be a bit slower than Taq, which is usually quoted as 1000 bases/min, so maybe increase this to 5 min. Don't worry about degrading the template/polymerase, as 72°C should not affect them too much in a PCR mastermix.
- Badges
- Science method