I am doing PCR amplification of 8.5 kb DNA fragment. I have observed a wierd result. When I use a master mix of 50 micL, I get the amplification but when I use 100 micL reaction it does not give any amplification. I am using Q5 High fidelity DNA polymerase. And I am using Touch Down PCR. Can anyone guide me in this regard. What is the possible reason?
large volume requires different time for each step. the best way to obtain high amount/volume of your product is to run a few reactions in lower volume .. instead of spending time to optimize large volume conditions....
Check that your thermocycler is rated for the larger volume. Many thermocyclers determine the temperature by calculating off of the volume and not by direct measurement. If the equipment is only rated for a smaller volume you won't be reaching the expected temperatures when you increase the reaction volume.
It could also be that you are simply not mixing the reactions as thoroughly at the larger scale too which would lead to poor amplification.
large volume requires different time for each step. the best way to obtain high amount/volume of your product is to run a few reactions in lower volume .. instead of spending time to optimize large volume conditions....
Take a look at how your tubes sit in your thermo cycler block.
Almost always only the lower portion of the tube is in the block, and a significant part of the upper (usually empty) portion of the tube is not in contact with the block.
If the fluid in your tubes is not contained completely in the portion within the thermoccyler block you will not be able to bring the liquid in the tube to set temperatures no matter how long your holds are. If some of it is in the portion of the tube siting in the air space between the block and the lid heater. You will set temperature gradients and currents with warmer liquid near the contact area rising as cooler liquid from above the block is dropping. I think a likely failure point in this scenario is never reaching adequate denaturation temperature.
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