I am using Invitrogen Plasmid Midiprep kit and I am wondering which buffer and what components allow the DNA to bind to the filtration column. Usually it is the Binding buffer that does this but for the kit I am using, there are only Equilibration Buffer, Resuspension, Lysis, Neutralisation and Elution buffer. So I am not sure which of these buffer acts as the Binding Buffer.
Also, in a binding buffer (or inthis case the buffer that acts as a binding buffer), what is the composition and what allows the DNA to bind to the filtration column?
Thank you in advance for providing me the answers.
The binding is due to the negatively charged DNA binding to a positively charged column, binding efficacy is mostly influenced by salt content and ethanol.
It is hard to say without knowing which kit you are using, there are many invitrogen kits, including some which have been discontinued. You should be able to find the protocol online for your specific kit. It is unusual that there is no wash buffer either, are you sure that all the buffers are there
Usually it is the neutralisation buffer which is needed for binding, this contains the salts needed to ensure binding. Without the protocol it is hard to say but the most likely protocol would be to pellet the cells and then re-suspend in resuspension buffer. Then add lysis buffer. Then add the neutralisation buffer (mixing immediately) and centrifuge to clear the lysate. Then add the lysate to the column to bind the DNA to the column. Discard flow-through and wash column before eluting in TE buffer or water.
With a bit more information about your specific kit I may be able to help further.
Hi, Willian,
Since you use the Invitrogen Plasmid Midiprep kit, I'll base my answer on that. Quoting from the users' manual, "PureLink HiPure Plasmid DNA Purification Kits" (https://www.fishersci.com/shop/products/invitrogen-purelink-hipure-plasmid-midiprep-kit-2/p-4926487, details included) use a patented anion-exchange resin (insoluble organic polymer containing cation groups that attract and hold anions present in a surrounding solution in exchange for anions previously held) ] to purify plasmid DNA to level equivalent to two passes through CsCl gradients The resin combines excellent capacity with fast flow rate, high resolution, high yield, and efficient endotoxin removal." I'm including a link to the manual. It details the composition of optimal buffers for washes and elutions and includes their composition.
Much luck on your experiment!!
Amy Jesionowski
Sam Amsbury,
Thank you very much for the information. The kit I am using is PureLink HiPure Plasmid DNA Purification Kits and Wash buffer is provided (I forgot to add this in my question). So the binding for a Midiprep kit works slightly different from a PCR Purification kit (this comes with a binding buffer)? The OCR purification kit I'm using is PureLink™ Quick Gel Extraction and PCR Purification Combo Kit from Invitrogen.
My Midiprep works but I want to know the principle behind it. I tried to google this but the search results are not very helpful. So I though maybe fellow scientists here can help me out with this.
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