Hi Lisa,
I'm following along here for best tips as well! We've had variable results with the kits that we have used. It's interesting that you say the PacBio Nanobind Nuclei kit did not work for you, that was going to be our next trial. I was really hoping the nuclei extraction would help. The kits we have used are the following:
-Machery Nagel Nucleobond HMW kit with extended to overnight lysis (this has variable results but has been working the best out of all we tried, it has CTAB in the lysis buffer )--we are not satisfied with this kit though and want to improve our success rate.
-MagMax Plant Extraction kit with PVP added. This one came at the recommendation of another core, but it did not perform well in our hands.
-Monarch HMW DNA purification kit. We really did not like this one. Sometimes the DNA would not even attach to the beads.
-Qiagen MagAttract HMW kit. This is our go-to kit for non-plant tissues, but alas it doesn't cut it for the plants and other notorious species.
As I mentioned, we've purchased the reagents to perform the nuclei isolation and Nanobind extraction but haven't had an opportunity to test it out yet.
I've found that the upstream handling by the client has a major impact on the success of the extraction, but unfortunately that is not something we always have control over.
I'm interested in what others have to say as well. For us so far, the Nucleobond has performed the best. I'm happy to help share any tips we have learned, so let me know if you have any specific questions!
Cheers,
Elizabeth
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Elizabeth Hudson
University of Louisville
Louisville KY
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Original Message:
Sent: 05-13-2024 16:37
From: Lisa McDougall
Subject: HMW gDNA from Plants?
Hello ABRF Colleagues,
Has anyone performed HMW gDNA extractions on plants, specifically blue mustard (similar to arabidopsis)? We have tried a couple kits, with poor results, and are wondering if anyone could recommend a kit or method that they've used before. We followed each protocol exactly as written i.e. not vortexing, mix by inversion, wide-bore tips etc. The two kits we've tried so far are PacBio NanoBind Plant Nuclei Kit and Promega Wizard HWM DNA Extraction Kit; we have a lot of experience with extractions, but nothing in the HMW realm. The end goal would be long-read sequencing.
Any suggestions and/or recommendations would be greatly appreciated!
Thank you,
Lisa McDougall
Research Associate | Molecular Research Core Facility
Gale Life Sciences Bldg 65 | Room 461
640 Memorial Drive Stop 8007 | Pocatello, ID 83209
Pronouns: She/Her/Hers
Idaho State University (Pocatello) acknowledges that it is located within the boundaries of the original Fort Hall Reservation on the traditional lands of the Shoshone and Bannock peoples.