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.
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.
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?
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.
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.
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.
Yes you can design a primer against the resistance gene
Thank you!
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.
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?
Good Video
Thank you
Please share my channel link with your friends and help me to reach big audiance
How can i avoid the smearing problem? What is optimum template concentration should use?
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
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.
Thanks for this vdo
Too good explanation
Please share it with your friends and help me to reach big audience
Can u explain how Ori of bacteria can be identified using PCR???
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 👍
subtitles are automatically generated in Japanese, can you replace them with English?
Oh I see ...I will do that
You missed magnesium's role