polymerase chain reaction ( PCR) | What are the 3 main steps in a PCR reaction? 

Please share it with your friends and help me to reach big audience

Could you please help me by sharing my contents with your friends group/ college group. I put huge efforts in making these videos but unfortunately not a lot of people are watching this.

Yes you can design a primer against the resistance gene

​@@animatedbiologywitharpanthank you for the great video. Can we get the slides for this for revision? Thank you very much.

Could you please help me by sharing my contents with your friends group/ college group. I put huge efforts in making these videos but unfortunately not a lot of people are watching this.

Please share my channel link with your friends and help me to reach big audiance

It's actually not simple. But trouble shooting cab be done by following ways. To avoid smearing , consider these steps: 1. **Optimize Primer Design:** Ensure your primers are specific to the target sequence and have similar melting temperatures. 2. **Template Quality:** Use high-quality DNA templates free from contaminants or degradation. 3. **Template Concentration:** Use an appropriate DNA template concentration. Too much template can lead to non-specific amplification. 4. **PCR Conditions:** Optimize annealing temperature and extension time to prevent non-specific amplification. 5. **PCR Cycling Parameters:** Follow manufacturer's recommendations for enzyme, buffer, and cycling conditions. 6. **Hot Start PCR:** Consider using hot-start enzymes or reagents to prevent non-specific binding during the initial setup. 7. **Gradient PCR:** If unsure about the optimal annealing temperature, perform a gradient PCR to identify the optimal temperature. 8. **Negative Controls:** Include negative controls (no template, water) to detect contamination or non-specific amplification. 9. **Template Denaturation:** Ensure proper template denaturation during the initial PCR cycle. 10. **PCR Cleanup:** Purify PCR products using methods like gel electrophoresis or commercial kits. 11. **Agarose Gel Analysis:** Analyze the PCR products on agarose gels to confirm the presence of the expected size band and absence of smears. 12. **Primer Dimers:** Minimize primer dimer formation by designing primers with appropriate melting temperatures. 13. **Template Source:** If working with complex samples, consider using purified DNA or additional purification steps. 14. **Troubleshooting:** If smearing persists, troubleshoot by adjusting annealing temperature, extension time, or reagents. Remember that optimization may require some trial and error. Regularly validate your protocol to ensure consistent and accurate results.

Okay got it. thank you

You just have to design specific primers against the ori sequence

@@animatedbiologywitharpan Thanku so much...... Any other requirement along with primer?

@@jabifatma2844 ya the buffer and primers has to be there together also optimal reaction temperature and MgCl2

@@animatedbiologywitharpan Thanku so much.... videos r helpful....I appreciate 👍

Oh I see ...I will do that