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Hi everyone,
this is the main discussion space for BOMB protocol #4.2 BOMB Clean-up and size exclusion using carboxyl-coated MNPs
Here you can post all your questions and feedback!I am interested in using this protocol with AMPure XP beads. My goal is to clean-up PCR reactions for Sanger sequencing at less cost and less hands-on time than clean-ups using silica columns.
The AMPure literature says that I should add ~2 volumes their beads to 1 volume of PCR reaction. Their literature also says that 5 uL beads is more than enough to bind any reasonable quantity of DNA.
So, my plan is clean-up my PCR reactions by adding 5 uL AMPure XP beads and ~2 volumes BOMB binding buffer (prepared without beads) to 1 volume PCR reaction (50 uL, typically). This protocol will allow me to reduce the amount of AMPure XP beads by 20-fold and thus save money.
Do you think my idea will work?
Do I need to “wash” the AMPure XP beads first? BOMB protocol 4.2 says that carboxyl-coated beads should be washed before preparing the binding buffer. What do you wash them in? The protocol gives no instructions for the wash step.
Thanks for your help!
Hi Hannah,
In general, the AMPure XP beads should work as well, with our buffer system, however, please keep in mind that they have a different structural composition than the BOMB beads. While the AMPure XP beads have a polystyrene core covered in a magnetite and a carboxyl layer, BOMB beads have a ferrite core and a carboxyl surface. Therefore, the beads differ in size and might exhibit different behaviour in the buffer system (especially with settling). In BOMB #6.3 we compared Sera-Mag Speed beeds (Silica-surface) with our silica beads and got slightly worse results with the Sera-Mag beads. This was also tried for plasmid extractions by a community member recently (see here). But I’d say go for it, give it a try and let us know, if it works for you and if yes, what yields [% input] you got 🙂
The amount [in ul] of beads to use depends strongly on the concentration of the beads. I think 5 ul is a reasonable starting point, if AMPure suggests this. But maybe give it a try with 2.5 ul and 10 ul as well, to figure out the optimum.
For the volume of binding buffer, it depends on the fragment size of your PCR product. If it’s larger than 200 bp, even 1.5 V (see exemplary results of #4.2) would be enough. But if your goal is to remove nucleotides, salts and proteins, anything between 1.5 V and 10 V should give similar results. As you don’t know how well the AMPure XP beads will perform, I’d start indeed with 2 or 3 V.
I don’t know in what liquid the AMPure XP beads are stored in. We include the washing step with water (thanks for pointing that out! We’ll include that in the next update of the protocol) to remove left over traces of the detergent used in the synthesis protocol (#3.1). So if the AMPure beads are stored in water, you should be ready to go.
I hope it works for you and let us know, if you have any more questions!
Cheers
Phil@hsseidel, we just uploaded two new community protocols that use commercial Sera-Mag beads with optimized BOMB buffers. Maybe they’ll help you increseyour yield as well 🙂 Look for #5.3 and #5.4 here (https://bomb.bio/protocols/)
Hi guys,
I have a very trivial question, sorry about that. I can’t seem to manage to seal my plates properly before vortexing during the washing steps. The 80% ethanol spills out of some of my wells, entering between the plate and the sealing foil. I tested two different types of foil, but both have the same problem, the plastic foil more and the aluminum foil less. I am pretty sure that I properly applied the foil because during PCR it seals perfectly. Maybe it’s because the ethonol does something to the glue?
Could you please tell me how you perform the washing step (vortexing or not) and what kind of foil or cover you use, if any? Do you use regular 0.2 ml PCR plates?
Thanks a lot in advance!
Hi Miriam – our group generally use deep well plates (1-2 mL) so when mixing using 1000 rpm on, for example, an eppendorf thermomixer, you won’t have to use seals at all.
Best wishes,
Tim
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Please in your protocol 2.1 you have mentioned in the first line:
<div>Combine your DNA sample with 5 µl stock solution of silica-coated magnetic</div>
<div>beads (BOMB protocol #2.1) and 2 volumes of binding buffer (for size exclusion</div>
<div>see Modification 1) in a 96-well PCR plate and mix until you receive a</div>
<div>homogenous suspension.</div>
<div>Question: What is the concentration of the silica coated magnetic stock solution ? also 2 volumes of binding buffer means 10 uL? Thank you</div>
<div>sorry for any inconvenience</div>
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