Hi guys, Welcome to the Advanced Video Search Engine, and you are watching How SPRI beads (SPRIselect, AMPure, etc.) work for nucleic acid clean-up & size selection. That video is uploaded by the bumbling biochemist at 2023-05-09T20:44:18-07:00. We are promoting this video only for entertainment and educational purpose only. So, I hope you like & enjoy it. [79cYJo_Cvqo]
| Name | How SPRI beads (SPRIselect, AMPure, etc.) work for nucleic acid clean-up & size selection |
| Video Uploader | Video From the bumbling biochemist |
| Upload Date | This Video Uploaded At 10-05-2023 03:44:18 |
| Video Discription | SPRI (Solid-Phase Reversible Immobilization) beads are used frequently in the preparation of DNA or RNA sequencing libraries. The protocols for library prep involve adding adapters, doing reverse transcription and/or PCR, etc. - steps that leave you with enzymes, primers, adapters, and other things you want to remove. So you need to do a number of purifications along the way to a library that’s ready to sequence. Thankfully, SPRI beads (SPRIselect, AMPure, RNAClean XP, etc.) make this easy(ish). blog: https://bit.ly/SPRIbeads The basic idea is: - take fragmented DNA (or RNA) - add paramagnetic beads (they’ll only act like magnets if you place them on a magnet) in a solution of PEG & NaCI - under those conditions, the DNA/RNA precipitates & binds beads - by modifying bead solution:sample ratio you can selectively bind sizes of interest - use magnet to capture the beads & pipet out other stuff - wash with ethanol while they're bound - add water or aqueous buffer to re-dissolve & elute How’s it work? - PEG & salt hog water, leaving less for DNA or RNA - PEG causes "crowding" that promotes the DNA or RNA sticking together - salt ions shield charges to prevent the strands repelling one another or the beads - the beads are coated with negatively-charged carboxyl groups that under "normal" conditions will repel DNA or RNA, helping it unstick The higher the PEG+salt vs. nucleic acid sample solution ratio, the smaller the DNA/RNA will precipitate (& bind beads). Only big stuff binds at a low ratio, but smaller binds too at higher ratios. You can do a low ratio to remove large (toss beads), followed by a high ratio one on the supernatant to select mid-size ones. This is referred to as a double-sided size selection. It works because it's harder to precipitate smaller DNA or RNA fragments than bigger ones. So, under milder conditions, you can selectively precipitate & remove big ones. - First get rid of big fragments - start with a low bead (and thus PEG & NaCI) to DNA/RNA solution ratio at which only big DNA pieces bind. - use a magnet to isolate & discard big-DNA/RNA-bound beads - Then wash off small fragments - add supernatant to more beads/PEG/NaCI - here, smaller pieces (but not tiny ones) bind too - wash while bound - Leaving medium-sized ones - add buffer to elute Sorry I don’t have much text (at least for now)! But I do have resources! Setting the stage…. You can size-selectively precipitate DNA with PEG/NaCl! Lis, J. T., & Schleif, R. (1975). Size fractionation of double-stranded DNA by precipitation with polyethylene glycol. Nucleic acids research, 2(3), 383–389. https://doi.org/10.1093/nar/2.3.383 You can use beads! DeAngelis, M. M., Wang, D. G., & Hawkins, T. L. (1995). Solid-phase reversible immobilization for the isolation of PCR products. Nucleic acids research, 23(22), 4742–4743. https://doi.org/10.1093/nar/23.22.4742 You can go bigger! Stortchevoi, A., Kamelamela, N., & Levine, S. S. (2020). SPRI Beads-based Size Selection in the Range of 2-10kb. Journal of biomolecular techniques : JBT, 31(1), 7–10. https://doi.org/10.7171/jbt.20-3101-002 The SPRIselect User Guide from Beckman Coulter has good figures, etc. https://research.fredhutch.org/content/dam/stripe/hahn/methods/mol_biol/SPRIselect%20User%20Guide.pdf Here’s a guide to making your own bead mixtures to save money - Solutions for purifying nucleic acids by solid-phase reversible immobilization (SPRI); Philippe Jolivet and Joseph W. Foley; Ludmer Centre for Neuroinformatics and Mental Health, October 21, 2015 - Based on DeAngelis MM, Wang DG, Hawkins TL, “Solid-phase reversible immobilization for the isolation of PCR products.” Nucleic Acids Res 1995, 23:4742–4743. https://s3-us-west-2.amazonaws.com/oww-files-public/f/f8/SPRI_buffers_v2_2.pdf and even if you go the pre-made route, this guide is still helpful at showing you what’s in it! Their recipes for DNA and RNA are: * DNA mix: 10 mM Tris base, 1 mM EDTA, 2.5 M NaCl, 20% PEG 8000, 0.05% Tween 20, pH 8.0 @ 25 °C * RNA mix: 1 mM trisodium citrate, 2.5 M NaCl, 20% PEG 8000, 0.05% Tween 20, pH 6.4 @ 25 °C And they use it with Sera-Mag™ Magnetic SpeedBeads™, carboxylated, 1 μm, 3 EDAC/PA5 (GE Healthcare Life Sciences #65152105050250) This review talks about a number of considerations and aspects of sequencing library preparation: Quail, M. A., Swerdlow, H., & Turner, D. J. (2009). Improved protocols for the illumina genome analyzer sequencing system. Current protocols in human genetics, Chapter 18, 10.1002/0471142905.hg1802s62. https://doi.org/10.1002/0471142905.hg1802s62 Finished in comments 79cYJo_Cvqo |
| Category | Science & Technology |
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