To accommodate the long product size (1kb-2kb), consider extending the extension time at 72 degrees in your PCR thermal profile.

Alternatively, you may perform a secondary PCR by using your initial PCR product as a template to further amplify the desired sequence.

Send yours PCR profile. it may be less then 1 min.

Increase their extension time upto two min or more.

I know it sounds counter-intuitive, but diluting out the DNA will often give you brighter bands. Lots of DNA preps will have PCR-inhibiting proteins or other molecules. Do a 1:10 and 1:100 dilution of a few samples and see if that helps.

One more query from my side why you are preparing 0.8% gel for 1-2kb.

I am giving you a brief.... may there be a one reason from this

Light bands in gel electrophoresis can be caused by various factors, and changing the master mix may or may not solve the issue. It’s essential to troubleshoot systematically before switching. Here are some common reasons for light bands and suggestions to improve the results:

1. Sample Quality

• Low DNA/RNA Concentration: Ensure your template DNA or RNA is sufficiently concentrated. If your starting material is too dilute, amplify more template or concentrate it before PCR.
• Template Purity: Contaminants in the DNA (e.g., salts, phenol, ethanol) can inhibit amplification.

Solution: Quantify your sample using a spectrophotometer (e.g., Nanodrop) and check purity ratios (A260/A280, A260/A230). Re-purify the DNA if needed.

2. PCR Reagents

• Master Mix Efficiency: Suboptimal master mix components, degraded Taq polymerase, or incorrect buffer conditions can result in weak amplification.
• dNTPs Degradation: Check that your dNTPs have not degraded.
• Magnesium Concentration: Suboptimal MgCl₂ levels can weaken amplification.

Solution: Try a fresh batch of master mix or prepare individual components yourself. If switching to a different master mix, ensure it is optimized for your template and primer design.

3. Primers

• Suboptimal Primer Design: Poorly designed primers may result in weak amplification or nonspecific products.
• Primer Degradation: Old primers may degrade over time.

Solution: Check primer design using online tools (e.g., Primer3 or NCBI Primer-BLAST). Use fresh primers if they’ve been stored for a long time.

4. PCR Conditions

• Insufficient Cycles: Too few cycles can result in low amplification.
• Annealing Temperature: Incorrect annealing temperature can reduce amplification efficiency.
• Extension Time: If the target fragment is long, insufficient extension time may lead to incomplete products.

Solution: Optimize PCR conditions (e.g., adjust annealing temperature ±2°C, increase the number of cycles, or extend the extension time).

5. Gel Loading

• Low Loading Volume: Insufficient DNA loaded onto the gel can result in faint bands.
• Staining Issue: Suboptimal staining with ethidium bromide or SYBR Safe can lead to faint bands.

Solution: Load more sample per well or increase DNA concentration. Ensure the gel is stained adequately or use a more sensitive stain.

6. Gel Electrophoresis Setup

• Low Voltage or Short Running Time: These can cause faint band visualization.
• Gel Concentration: 0.8% gel is appropriate for larger fragments but not for very small ones.

Solution: Verify the electrophoresis setup and run conditions. Ensure proper gel and buffer composition.

7. Master Mix Specificity

If none of the above resolves the issue, consider switching to a high-fidelity or hot-start master mix, as these can improve specificity and yield. Before switching, consult the master mix’s datasheet and compare features.