CTAB - Chloroform DNA Extraction from Fungal Tissue - Bonito Lab Training
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- čas přidán 20. 12. 2020
- Virtual training for how to isolate high-quality DNA from fungal tissue using the CTAB - chloroform method in the Bonito Lab at Michigan State University. Produced, filmed, and narrated by Julian Liber.
*Correction: At 11:04, centrifugation time should be 60-90 seconds with 80% ethanol.
The protocol is adapted from Doyle 1991. scholar.google.com/citations?... - Věda a technologie
Thankyou sir ❤
awesome 👍🏻
Please upload a PCR amplification video too
Hello sir.. I’m working on microspodian spores ( a sister fungus). Nosema are endoparasites of honey bees. Spores are of nearly 2 microns of size. Also they only exist in the abdomen of bees. For grinding purpose I usually take few bees and properly grind them in lysis buffer. I used commercial kit for DNA extraction but I didn’t get DNA of those microscopic spores.
As I didn’t see bands on electrophoresis. I don’t know where I mistaken.
As I didn’t get band even of honey bee’s DNA. 😒
How about this method? Would it help me?
What else Should I do?
Plz help me. It’s a request
There are a few procedures to try which may improve your DNA yield. You can grind your tissue in a mortar and pestle with liquid nitrogen to make a very fine powder. This may help break open the spores before your add the lysis buffer. You may also want to process more samples, where you would suspend the pellets in a smaller amount of water (~20 uL) then combine. Other kits or extraction protocols may be more suitable than this one for your sample type.
@@julianliber4633 u mean I am making a mistake at grinding! Probably my homogenate is not well ruptured to release the cellular contents..
@@SaumyaSharma123 it is possible, but I think that is often a problem when working from spores instead of hyphae.
@@julianliber4633 okok.. I used DNeasy plant mini kit and followed the procedure. I didn’t get anything on electrophoresis. Lets see how this CTAB technique will work!
How do u prevent scraping agar while removing mycelium from plate
The fungi im working on has very flat colony
You are Godsend. Thank you so much. By the way, do you have a citation for your source of protocol? thank you
I just added it to the video description.
Hi!!
This is a very well explained protocol, in your video you mention that a more stringent protocol is needed for nanopore sequencing, I will be doing mine with nanopore do yo have a protocol to follow with the description for such extraction? Thanks in advance
You will likely have to optimize your protocol based on the organism you are working with. This page has some protocols which have been successful for producing high molecular weight DNA for PacBio or Nanopore sequencing 1000.fungalgenomes.org/home/protocols/high-quality-genomic-dna-extraction/
@@julianliber4633 Thank you so much this is very much appreciated, keep up with the great work :D
@@lauraesquivel1019 Just found another protocol which you may find useful: dx.doi.org/10.17504/protocols.io.qtjdwkn
Thanks for this video. I want to ask is there any need to make the mentioned solutions of specific molarity? If yes, then let me know the molarities of solutions used here. My 2nd question is, has this method given better results so far if anyone has used this method?
I believe that the CTAB buffer may be the only one. The recipe can be found here: drive.google.com/file/d/1yt6WUtvR5n-fI4a2NkXBlq9d3S08JxQx/view?usp=sharing
This protocol provides DNA of good enough quality for Illumina sequencing (HiSeq, NovaSeq), but is not of sufficient quality for Nanopore or PacBio.
Hi, great video :)
I'm new to this. How did you manage to scrape the fungal tissues away from the agar so easily? Mine always tear up.
We often place sterilized cellophane sheets over the agar surface. You can cut the cellophane to the size of the plate bottom, then place in water and autoclave. Needs to be cellophane, not another kind of plastic, so that it can withstand autoclaving and allows for nutrients to pass through.
@@julianliber4633 Thanks for the fast reply! ^_^
Is there a particular grade or specific name of the cellophane - I can't seem to find more info online on that, and where to get it?
The cellophane is placed on top of the agar and the fungal tissues will grow on the cellophane instead of agar?
@@lwwan5935 Yes, the idea is that the tissue will grow on top of the agar. As for the cellophane, a product like this should work. www.amazon.com/dp/B08XXLJV9T/ref=cm_sw_r_cp_apa_glt_fabc_82RKBF225HFC7601KJTY
@@julianliber4633 I see. Great! Thanks a lot for your help! ☺☺
@joshuamc Thanks for the suggestion 👍👍
jai besion cette védio en français ....ou je peux trouvé
Can we use broth instead of agar plate for growing the fungal tissue?
Yes, just use a sterile spatula or filter to remove liquid from the tissue before extraction.
Would u like to give me the literature or journal of CTAB Chloroform extraction please? i need it for analyzing active content of defoamer chemical
It's adapted from Doyle 1991. scholar.google.com/citations?view_op=view_citation&hl=en&user=pMyR8RUAAAAJ&citation_for_view=pMyR8RUAAAAJ%3A35r97b3x0nAC
Sir please give the literature refrence. I want to add this protocol in my theisis, becuase its give best result.
It's adapted from Doyle et al 1991; scholar.google.com/citations?view_op=view_citation&hl=en&user=pMyR8RUAAAAJ&citation_for_view=pMyR8RUAAAAJ:35r97b3x0nAC
You can find the text here: link.springer.com/content/pdf/10.1007/978-3-642-83962-7.pdf
Can you suggest a simpler method? I just need to amplify ITS region.
This video has a faster method. For some yeasts you can skip extraction and add directly to the PCR with a longer initial denaturation. Buffer recipe is in the comments. czcams.com/video/pUgdz_gJvsg/video.html
Do you usually get pure and intact DNA from fungi with this protocol ? I am using a modified Promega protocol and yielded low quality DNA from most of my sample (degraded DNA and low A260/A230 ratios)
It really depends on the organism. This provides quality DNA for Illumina sequencing, but may not produce molecules with a high-enough molecular weight for ONT or PacBio sequencing.
@@julianliber4633 I will send them to Macrogen in Netherlands for sequencing. I don't know what kind of sequencing they use. Do have any suggestions for isolating good quality DNA from fungi (a better protocol for example)?
@@wick3109 it depends on which service you request. It looks like they do both Illumina and PacBio for whole genome sequencing www.macrogen-europe.com/services/next-generation-sequencing/whole-genome-sequencing
@@julianliber4633 OK thank you very much! I will just sequence ITS‚ GAPDH and TEF genes.
@@wick3109 you might be able to use this protocol if you are only doing PCR with the sample. czcams.com/video/pUgdz_gJvsg/video.html
I was rewatching this video and I just realized at 11:05 you said centrifuge for 90 MIN. instead of 90 seconds... Oops lol.
Didn't mean that! I can probably fix it in the captions and add a note. Thanks!
Is the isopropanol ice cold or room temparatyre
@@tawseefalaff9976 I typically chill it for this protocol, but if it's at room temperature then you can likely put it in the freezer for 1 or 2 minutes longer.
@@julianliber4633 Thank you so much
Speak slowly can makes this video better.
Your speaking speed is fast.
Please speak slowly and clearly. You are obviously reading from a protocol, and you sound very bored. I can't understand 80% of what you're saying.
What??? It was really simple and clear
bruh you can literally press shift + < to reduce the speed.