I need to make this reaction (pictured) with the same final concentration, but it needs to be in a total volume of 7.5ul instead of 25ul. Can someone please help me by walking me through your math process? I'm having a hard time figuring this out and would greatly appreciate any help.
This is just the Mastermix of the PCR reaction. There are no forward or reverse primers in this mix.
at 7.5ul you may find evaporation is a problem even with heated lids on the pcr machine. You will probably have to test parameters again at this small volume so you may want to think of oil overlay of the pcr reaction or just using a larger pcr volume and taking a suitable size aliquot for whtever the next stage of your process is
Well, decreasing the volume of everything by 1/4 will give you that total volume of 7.5 microliters.
But, it's very challenging to accurately and consistently measure volumes that small.
Why are you trying to get the volume so low? In general, PCR works better in larger reactions (20-50 microliters is common).
The "add water to make it 25 microliters" is the flexible part of this protocol. That's where you have the space to account for the volume of your primers & template/DNA.
I'd suggest talking with your supervisor to make sure you understand the protocol & how it fits into your overall experiment. Good luck!
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