Amplifying 8 targets in a single multiplex-PCR reaction can be challenging, as the success of the reaction is influenced by a number of factors such as the complexity of the target DNA, the size and number of amplicons, the specificity of the primers, the annealing temperature, and the concentration of the reagents. Here are some suggestions that may help you improve the yield of your multiplex-PCR reaction and maximize the chances of obtaining all 8 products:

1. Optimize reaction conditions: Start by optimizing the reaction conditions for each target separately, including the annealing temperature, extension time, and MgCl2 concentration.

2. Choose appropriate primer concentrations: It's important to use the correct primer concentration to ensure proper amplification. If the primer concentration is too low, the reaction may be inefficient, and if the concentration is too high, the reaction may produce non-specific products.

3. Avoid primer-dimer formation: Primer-dimer formation can lead to reduced yields or absence of specific products. To minimize this, use primers with high specificity, and avoid primer pairs that have complementary 3' ends.

4. Use appropriate template concentration: The concentration of the template DNA should be optimized for each target. If the template is too diluted, the yield may be reduced, while high template concentrations can cause smearing or non-specific amplification.

5. Consider using a touchdown PCR protocol: Touchdown PCR involves gradually reducing the annealing temperature over several cycles, which can help to reduce non-specific binding and improve specificity.

6. Monitor the reaction by gel electrophoresis: Visualizing the PCR products on a gel can help to identify potential issues with the reaction, such as primer-dimer formation or smearing.

7. Try increasing the number of PCR cycles: Increasing the number of cycles can help to increase the yield of the reaction, but be careful not to over-amplify the DNA, as this can lead to increased non-specific products.

8. Consider using a high-fidelity polymerase: High-fidelity polymerases can help to reduce non-specific products and improve the yield of the reaction.

9. Consider using a proofreading polymerase: Proofreading polymerases can help to reduce errors during the amplification process, which can improve the accuracy of the final products.

I hope these suggestions will help you optimize your multiplex-PCR reaction and improve the yield of your 8 target products. Good luck!

Mansoor Khan Thanks! It really helps me.

Mansoor Khan

Have you ever heard chemical or physical methods to enhance the successful rate of multi-PCR

As of my knowledge , there are various methods employed to enhance the success rate of multiplex polymerase chain reaction (multi-PCR), which is a technique used to simultaneously amplify multiple DNA fragments in a single reaction. These methods can be broadly classified into chemical and physical approaches.