Do you happen to know any canadian places that will do sequencing, without requiring full 96-cell plates or belonging to their university? I'd love to try this out, but i'm not sure what my options are for sequencing ~10 samples at a time in a more consumer-friendly fashion. thx for all your great videos!
I need to amplify large segments of upto 10-12 kb. It says the method is not suitable if we want unfragmented DNA. I needed to know the extent of fragmentation and whether I can still consider this method for my purpose.
Dear Netra, our protocols that we use the HotSHOT DNA Extraction Kit for - and the literature we've read online - only require short-read DNA so we have not tested the maximum length of DNA fragments that can be obtained by this method. That's not to say it wouldn't work, but we couldn't guarantee you could get 10-12 kb reads using it.
Yes, and if you want to store the resulting sample long-term, it's a good idea to purify the DNA from the HotSHOT kit as it's a rough method, so otherwise the final extract will degrade over time. How do you want to use it once purified - what's the end goal? The caveat with HotSHOT is that it fragments the DNA, so you'll get PCR grade DNA, but not samples suitable for total DNA analysis.
@@BentoLab So... I tried to purified genomic DNA (from chicken breast for the test) but I found a major peak at 220 nm in the UV spectra. I tried several times. Protocole : after neutralisation, I added the equal volume of isopropanol, mixed gently and centrifuge at max speed. Then two 70° etOH wash, and dilution of final pellet with ultrapure water. Any idea regarding this 220 nm peak? 🙄
@@dendro-tv Hi there, we are not sure what's happened there. Proteins absorb UV light at 220 nm so if they are causing the peak that suggests your purification has not been successful. This week we've been investigating PCR inhibition in DNA extractions done using the HotSHOT kit and have had much greater PCR success when using a) 1 in 10 dilutions of the DNA extractions in water, and b) top fraction of partially thawed DNA extractions (either an effect of gravity or defrost rate of different molecules - we're not sure). If you are looking to purify the extractions for long-term storage, then we would suggest 1 in 10 dilutions of the DNA extractions in water or TE buffer as an alternative solution.
Good Q. About 1-2mL should be enough, but it's more about the size of the pellet of cells. Check out the protocol here, as it gives a guideline on pellet size: bento.bio/protocol/biotechnology-101/dna-extraction-from-saliva/
Do you happen to know any canadian places that will do sequencing, without requiring full 96-cell plates or belonging to their university? I'd love to try this out, but i'm not sure what my options are for sequencing ~10 samples at a time in a more consumer-friendly fashion. thx for all your great videos!
Is that possible to extract HPV viruses from urine sample/swab sample for RTPCR test?
Can you extract the DNA from a feather?
Yes, check out our online protocol bento.bio/protocol/bird-sexing/
I need to amplify large segments of upto 10-12 kb. It says the method is not suitable if we want unfragmented DNA. I needed to know the extent of fragmentation and whether I can still consider this method for my purpose.
Dear Netra, our protocols that we use the HotSHOT DNA Extraction Kit for - and the literature we've read online - only require short-read DNA so we have not tested the maximum length of DNA fragments that can be obtained by this method. That's not to say it wouldn't work, but we couldn't guarantee you could get 10-12 kb reads using it.
Hi! Do you know if a genomic DNA precipitation/purification with isopropanol is still possible after HotShot lysis?
Yes, and if you want to store the resulting sample long-term, it's a good idea to purify the DNA from the HotSHOT kit as it's a rough method, so otherwise the final extract will degrade over time.
How do you want to use it once purified - what's the end goal? The caveat with HotSHOT is that it fragments the DNA, so you'll get PCR grade DNA, but not samples suitable for total DNA analysis.
@@BentoLab just PCR. Yes I would like to store sample for a long time in freezer. 😉
@@dendro-tv Great! Give us a shout if you have other Qs when you try it out.
@@BentoLab So... I tried to purified genomic DNA (from chicken breast for the test) but I found a major peak at 220 nm in the UV spectra. I tried several times.
Protocole : after neutralisation, I added the equal volume of isopropanol, mixed gently and centrifuge at max speed. Then two 70° etOH wash, and dilution of final pellet with ultrapure water. Any idea regarding this 220 nm peak? 🙄
@@dendro-tv Hi there, we are not sure what's happened there. Proteins absorb UV light at 220 nm so if they are causing the peak that suggests your purification has not been successful. This week we've been investigating PCR inhibition in DNA extractions done using the HotSHOT kit and have had much greater PCR success when using a) 1 in 10 dilutions of the DNA extractions in water, and b) top fraction of partially thawed DNA extractions (either an effect of gravity or defrost rate of different molecules - we're not sure). If you are looking to purify the extractions for long-term storage, then we would suggest 1 in 10 dilutions of the DNA extractions in water or TE buffer as an alternative solution.
OMG so cool
Thanks!
How much of a saliva sample would I need for each tube?
Good Q. About 1-2mL should be enough, but it's more about the size of the pellet of cells. Check out the protocol here, as it gives a guideline on pellet size: bento.bio/protocol/biotechnology-101/dna-extraction-from-saliva/