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

[@frederickpollmeier1861]

Good Video

[@AmitThakur-py4te]

Hello brother i am your very old follower. How to identify resistance gene using pcr after antibiotics resistance profile. Gene sequence is like available at ncbi we can design primer from primer blast.

[@animatedbiologywitharpan]

Yes you can design a primer against the resistance gene

[@ps-we9dt]

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

[@deepikamks]

Thanks for this vdo

[@homaehsan2844]

hello, I have a question regarding PCR and non-specific amplification. we see a band in our negative control ( which is water), Do you think we can explain it as a non-specific amplification? we have changed everything and the suspicion for having contamination is low. what do you think?

[@samantadm8269]

Thank you!

[@animatedbiologywitharpan]

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.

[@pratitidas]

How can i avoid the smearing problem? What is optimum template concentration should use?

[@animatedbiologywitharpan]

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.

[@pratitidas]

Okay got it. thank you

[@anushabhat4135]

Too good explanation

[@animatedbiologywitharpan]

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

[@ReanC]

Thank you

[@animatedbiologywitharpan]

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

[@gothamibandara5607]

thank you very much

[@animatedbiologywitharpan]

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.

[@jabifatma2844]

Can u explain how Ori of bacteria can be identified using PCR???

[@animatedbiologywitharpan]

You just have to design specific primers against the ori sequence

[@jabifatma2844]

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

[@animatedbiologywitharpan]

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

[@jabifatma2844]

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

[@devjotimondal6410]

You missed magnesium's role

[@ДмитрийГолубев-ю7у]

subtitles are automatically generated in Japanese, can you replace them with English?

[@animatedbiologywitharpan]

Oh I see ...I will do that