you could try running an annealing temperature gradient to find a higher annealing temperature and then run samples with added dmso at 1% up to 8% to clear up secondary structure problems in case your template has significant secondary structure and if these fail then run touchdown pcr. If everything fails than redesign the primers

What is the length of your designed primers? I suggest you first design primers without hang regions and then amplify them. After you get bands then extract the bands from gel and again run by NdeI (forward primer) and HindIII as a reverse primers. you will get results. Use PCR condition as :

94 for 4 mint

94 for 30 sec

55 for 30 sec

72 for 1:30 mint

72 for 10 mint

with 35 x cycles

Zinc finger proteins are among the most abundant proteins in eukaryotic genomes. Their functions are extraordinarily diverse and include DNA recognition, RNA packaging, transcriptional activation, regulation of apoptosis, protein folding and assembly, and lipid binding

If you are not obtaining the desired band for your zinc finger protein 179, and you are observing unexpected bands on the agarose gel, several troubleshooting steps can be considered to optimize your PCR reaction. Here are some suggestions:

1. Primer Design:

• Check Primer Sequences:Ensure that the primer sequences are correct and do not have any unintended matches in the genomic DNA. You might want to double-check the specificity of your primers using bioinformatics tools.
• Optimize Primer Concentrations:If necessary, optimize the concentrations of the forward and reverse primers in your PCR reaction. Too much or too little primer can impact the specificity and efficiency of the reaction.

2. PCR Conditions:

• Adjust Annealing Temperature:Optimize the annealing temperature in your PCR program. Use a gradient PCR or try different temperatures to find the optimal one for your primers.
• Optimize Extension Time:Ensure that the extension time is appropriate for the length of the desired product. Extending the time may be necessary for a longer product.
• Check MgCl2 Concentration:The concentration of MgCl2 in the reaction can affect specificity. Optimize MgCl2 concentration within the recommended range.

3. Template DNA:

• Quality of DNA Template:Ensure the quality and integrity of your template DNA. Contaminants or degraded DNA can lead to unexpected bands.
• Amount of Template:Optimize the amount of template DNA. Too much or too little DNA can affect the efficiency and specificity of the reaction.

4. PCR Program:

• Check Denaturation Temperature:Ensure that the denaturation temperature is sufficient for complete DNA denaturation.
• Increase Cycle Number:If necessary, consider increasing the number of PCR cycles to enhance the amplification of the target DNA.

5. Gel Analysis:

• Purify PCR Product:If you observe multiple bands, purify the PCR product using a gel extraction kit to remove unwanted fragments.
• Sequence the Bands:If you still encounter unexpected bands, consider sequencing them to identify their origin. It's possible that they may be nonspecific amplifications or artifacts.

6. Troubleshooting Unwanted Bands:

• Non-Specific Amplification:Unwanted bands may be due to nonspecific amplification. Refine primer design or consider using a hot-start polymerase to reduce nonspecific products.
• Contamination:Ensure that there is no contamination in your reagents or on your equipment. Use separate areas for DNA preparation, PCR setup, and gel electrophoresis.

7. Consider Alternative Enzymes:

• If the issues persist, consider using alternative restriction enzymes for cloning purposes.

8. Consult Colleagues or Experts:

• Seek advice from colleagues or experts in your field who may have experience with similar PCR reactions.

By systematically troubleshooting these aspects of your PCR experiment, you should be able to optimize the reaction conditions and obtain the desired band for your zinc finger protein 179. If the problems persist, consider consulting with experienced colleagues or reaching out to experts in the field for additional guidance.