I used a established PCR kit to amplify the target DNA. The instructions from kits was given to use (1.5-15ng/ul) concentration of DNA. After DNA extraction procedure i got 23ng/ul;then i adjust the required concentration through dilution. But after giving 10ng/ul DNA template into ready to use master mix, now the reaction volume is 30ul (20ul master mix +10ul DNA template). My question is the actual concentration of 10ng/ul of DNA will change after adding to the master mix? If the master mix contain any PCR grade water, the template will be diluted?
It depends on what your master mix protocol says.
There should be mentioned the final concentration of reagents needed, including your template DNA. If not, then just follow the instructions and don't be bothered by the final concentration you get after adding your template to master mix.
Also I suggest to recalculate your template concentration to absolute quantity. For example, in your case protocol requires 15 to 150ng of your template per reaction. Usually, when using master mixes, that's the one thing you need to worry about.
Hope that helps.
10ng/ul x 10ul = 100ng, DNA your provide in the reaction
total solution 30ul
DNA concentration = 100ng/30ul = 3.33ng/ul
Yes, DNA concentration will be diluted because the solution you add to your DNA template doesn't contain any DNA, no matter what kind of solution it is.
On the other hand, if you have two DNA templates in your reaction, the TOTAL final DNA concentration in the reaction is still 1.5~15ng/ul.
What is the brand of your Mastermix? Generally, the Mastermix ready to use are concentrated 2x, they contain the TAQ Polymerase, dNTP and MgCl2 as co-factor of the TAQ Polymerase. So depending on the desired final volume, you put half of Mastermix, 1/10 of the final volume of the two primers (10 pmoles), 1/20 of the final volume of the DNA template and nuclease free water to adjust the volume.
For example, if the final volume is 20 µL, you put 10 µl of mastermix 2X, 2 µL of each of the primers (2 µL forward and 2µL reverse), 1 µL of DNA 10 ng/UL and 5 µL of nuclease free water.
If you put 10 µL of your DNA, you will obtain a smear.
Typically, recipes will give a volume and amount of DNA to add. Like 1-2 ul of DNA of 5-100 ng. Can you post a copy of the protocol you are using? That would be helpful.
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