If you're facing challenges with the second phase of your overlapping PCR, where the Left/EGFP and Right/EGFP combinations are not amplifying, you can try the following;

1. If you already know the optimum primer concentration and Double-checked the primer sequences for Left, EGFP, and Right you might want to confirm that the primer binding sites do not contain any undesired secondary structures, such as hairpins or primer dimers.

2. Check the concentration of your template DNA. Too much or too little template DNA can impact the success of PCR. This could also help you optimize your Primer concentration

3. Consider optimizing the MgCl2 concentration by performing a gradient PCR. Different primers may require adjustments to these components.

4. Check the extension time in your PCR program. If the second phase involves a longer sequence, you may need to extend the elongation time accordingly.

5. Purify your PCR products before using them as templates in the second phase. Contaminants from the first PCR may interfere with the second round and also consider diluting the first PCR product before using it as a template for the second PCR. Concentrated PCR products may inhibit the second PCR reaction.

6. Repeat a mock experiment with the addition of PCR enhancers or additives like DMSO or betaine. You might just be lucky because these can sometimes improve PCR efficiency.

By systematically troubleshooting each of these factors, you should be able to identify the issue causing the lack of amplification in the second phase of your overlapping PCR. Remember to perform controls and consider running a gradient PCR to optimize conditions for both primer pairs.

Wishing you all the best in your present and future projects,

Warm Regards.