Its true that we prefer using nucelase free water. Its serves the purpose of maintaining the final volume. This is one component which increases the risk of nuclease contamination in your PCR reaction mixture so it is better rather essential to have nuclease-free water.
Thanks for asking this question. I had the same doubt. Further, I was wondering if the sole purpose of adding water is to make up the volume of the final reaction, can we eliminate this addition and instead add say, more template (keeping the volumes of the rest of the components of the master mix the same)?
To dilute the other reagents to their correct concentrations. Water is not added if the final volume places each reagent at the correct final concentration, which should be 1X or some other recommended concentration in moles. E.g., the buffer is likely at 10X stock. Your reaction should be at 1X. Once you add all the other reagents, you need to adjust the volume to produce the final desired 1X buffer concentration.
we must use nuclease free water in PCR reaction so as to prevent degredation of DNA.
Nuclease free water is used in order to dilute the concentration of the reagents to the proper final concentration. Also use of nuclease free water helps avoid DNA degradation by nucleases as well as interference of the PCR reaction by ions which could be present in otherwise not nuclease free deionized water.
Thank you all for your answers. I was looking for this question today and thanks to you I understood.
Nuclease free water basically provides a suitable medium for your amplification reaction.
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