PAGE gel extraction in action - a practical look at gel extraction using the crush & soak method скачать в хорошем качестве

PAGE gel extraction in action - a practical look at gel extraction using the crush & soak method

The crush & soak method is a technique you can use to extract DNA or RNA from polyacrylamide (PAGE) or agarose gels. This allows you to purify different size fragments, since the gel part separates those fragments by size. Problem is, the gel gel electrophoresis leaves them trapped in a gel mesh. So your job with gel extraction is to free the fragments you want! And one way to do this is the crush & soak method, which basically uses diffusion to let the fragments wander randomly out of the gel and into the nice clean liquid it’s soaking in. The crushing (and freezing) part helps the fragments more easily find their way out. Diffusion works best with big volumes. But you typically don’t want want to keep your DNA or RNA in huge volumes super dilute. So after diffusion does its thing (e.g. overnight) you can precipitate the fragments out w/alcohol, wash, & resuspend into a smaller volume. for more on the theory: blog: https://bit.ly/gel_extraction_crush ; YouTube:    • Gel extraction with the "crush and soak" m...     Note: there are easier ways with agarose since you can heat it and melt it. Especially if you use low melting point agarose. But PAGE gels don’t de-gel like that. So this way is still used a lot. Or electroelution which I’m trying to learn more about… Sorry I don’t have text written for this yet but here’s an outline and some links to more run a gel to separate the fragments by size. more here: blog form: http://bit.ly/ureapage ; YouTube:    • Nucleic acid PAGE (Polyacrylamide Gel Elec...     use agarose gel for big stuff, polyacrylamide (PAGE) for small stuff - can do non-denaturing to keep strands together or denaturing (e.g. urea-PAGE) to keep single-stranded things single-stranded & unfolded visualize the separated fragments can use fluorescent dye like SYBR Gold or UV shadowing (uses intrinsic UV absorbance). More here: http://bit.ly/fluorescentstains minimize UV exposure (of you and the gel) cut out band(s) of interest w/clean razor(s) or scalpel(s) minimize gel amount & be careful not to into bands you don’t want use a clean blade for each band & carefully dispose of blades in sharps waste container place each band in a separate nuclease-free microcentrifuge tube crush by smushing against the wall with a P1000 pipet tip this increases the surface area Add elution buffer this is nothing fancy just some pH-stabilized salt water with EDTA & maybe SDS. Its main role is just giving you volume for your fragments to escape into Freeze Stick on dry ice for 30 min Thaw This freeze-thaw cycle uses ice crystals to help break up the gel Thaw on a rotator for a long time (e.g overnight) - required time will depend on fragment size (longer ones are more tangled up in the gel & move slower & take longer) Isolate from gel bits e.g. with Costar SpinX tubes Precipitate out with salt+alcohol (EtOH or IsOH). much more on this here: https://bit.ly/na_precipitation; YouTube:    • DNA & RNA precipitation with alcohols & sa...   Can add a carrier like GlycoBlue to help Centrifuge & remove supernatant (the liquid) Wash off extra salts with 70-80% EtOH and remove liquid Air dry Resuspend on desired buffer Purification of oligonucleotides using denaturing polyacrylamide gel electrophoresis. Jack Pollard. 08/31/98 https://molbio.mgh.harvard.edu/szosta... BiteSizeBio. Yevgeniy Grigoryev, Crush Like an Elephant, Soak Like the Rain: Old-School DNA Gel Extraction. May 2013 https://bitesizebio.com/13534/crush-l... CSH Protocols. Isolation of DNA Fragments from Polyacrylamide Gels by the Crush and Soak Method. Michael R. Green and Joseph Sambrook. 2019 http://m.cshprotocols.cshlp.org/conte...      more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 http://bit.ly/2OllAB0 or search blog: http://thebumblingbiochemist.com