5 Tips for Successful PCR Reactions

In this post, I want to share with you about 5 tips for successful PCR reaction since we often fail in conducting the PCR reactions. I believe this PCR method is easy to be conducted by anyone, but the handiness of the person is very required. Here are those 5 tips:

1. You have to always store the ingredients, especially dNTPs and enzyme, in the -80oC freezer (if possible)for storage and on ice when you setting up the reactions. dNTPs and enzyme are two components of PCR reaction that are least stable. For practical use, you can aliquot the dNTPs and enzyme into several small tubes, enough for 50 or so reactions per tube and store them at -80oC. By this way, you only need to thaw a small amount for immediate use, and the rest are still kept in the freezer.
2. Use the buffer that comes with the enzyme. I mean if you use one brand of enzyme, and another brand of buffer, the reaction may not work as it has to be.
3. Add the enzyme to the Master Mix Last. Mix the Master Mix just before adding the enzyme, and again just after since the enzyme usually comes in a very viscous, dense buffer and it will sink to the bottom of your Master Mix, so it is very important to mix it well before delivering aliquots into reaction wells.
4. You have to make sure the thermocycler program is correct for the primer that you are using.
5. Also, make sure you are using the appropriate amount of Mg. The optimal concentration of Mg must be determined empirically for each set of primers. If you use published primer, usually the Mg concentration had been determined in the publication.

Hopefully these 5 tips will be useful for your PCR lab-work.