3:37 You only mentioned ethanol here. The protocol I have suggests an ethanol(90ml)-AL(86ml) mixture. Thanks for the video though. For someone like me who never worked in a lab, it was very useful.
My lab is worried about splash risk while working with field-collected mosquitoes that may contain human pathogens, so pestle homogenization is not ideal for me. Would 3mm stainless steel beads work fine?
Thanks for video! It has helped me a lot. I just have one question. I noticed that in the DNA precipitation step, you didn't use the AL buffer as is described in the manual of the product. So, is that buffer optional or just for specific samples?
Great question. Buffer AL is categorized as a lysis buffer, so we added it at 2:47 during the "Cell Lysis and DNA Precipitation" portion of our protocol. Here's a link to the protocol we use, which uses the DNeasy kit for insect extraction: www.qiagen.com/us/resources/resourcedetail?id=cabd47a4-cb5a-4327-b10d-d90b8542421e&lang=en I hope that helps, please let us know if you have further questions! Also feel free to email us at wolbachiaproject(at)vanderbilt(dot)edu.
Yes, Wolbachia can be detected in legs as reported here: www.ncbi.nlm.nih.gov/pmc/articles/PMC3342236/. Most of our participants isolate from fresh samples, but some have successfully amplified DNA from from dead organisms. Make sure to indicate the state of your organism along with any reported data.
This protocol is optimized with a shorter incubation time for classroom use. If you reduce the amount of proteinase K, we recommend increasing the incubation time to enhance lysis. Also make sure to efficiently grind the sample with micropestles.
3:37 You only mentioned ethanol here. The protocol I have suggests an ethanol(90ml)-AL(86ml) mixture. Thanks for the video though. For someone like me who never worked in a lab, it was very useful.
Thankyou so much ❤️
This was very helpful!
We love to hear that, thanks!
My lab is worried about splash risk while working with field-collected mosquitoes that may contain human pathogens, so pestle homogenization is not ideal for me. Would 3mm stainless steel beads work fine?
Thanks for video! It has helped me a lot. I just have one question. I noticed that in the DNA precipitation step, you didn't use the AL buffer as is described in the manual of the product. So, is that buffer optional or just for specific samples?
Great question. Buffer AL is categorized as a lysis buffer, so we added it at 2:47 during the "Cell Lysis and DNA Precipitation" portion of our protocol. Here's a link to the protocol we use, which uses the DNeasy kit for insect extraction: www.qiagen.com/us/resources/resourcedetail?id=cabd47a4-cb5a-4327-b10d-d90b8542421e&lang=en
I hope that helps, please let us know if you have further questions! Also feel free to email us at wolbachiaproject(at)vanderbilt(dot)edu.
i have one question. can we preserve whitefly in 70% Ethanol and extract DNA after 2 months? is it possible?
Can we extract the DNA from the legs of insects? And do we need to get a fresh sample or alive insects for DNA extraction
Romario, its good that u also watched this video.😄
@@saranga6304 🤣🤣🤣🤣
Yes, Wolbachia can be detected in legs as reported here: www.ncbi.nlm.nih.gov/pmc/articles/PMC3342236/. Most of our participants isolate from fresh samples, but some have successfully amplified DNA from from dead organisms. Make sure to indicate the state of your organism along with any reported data.
We Didn’t get dna. All lines we followed except 200 protinase K. We used 30 protinase K. Among 8 samples, we got for two.
This protocol is optimized with a shorter incubation time for classroom use. If you reduce the amount of proteinase K, we recommend increasing the incubation time to enhance lysis. Also make sure to efficiently grind the sample with micropestles.