@sebastian
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Hi,
I am not sure what you mean by your request, as all plasmid isolation protocols that involve Sera-Mag beads, do not involve guanidine salts.
Guanidine salts don’t play nicely with the Sera-Mag beads, this is why in all protocols that I provided with Sera-Mag you will not find any guanidine salts as components of the buffers.
Best regards,
Sebastian
Hi,
My personal experience (especially with SeraMag Beads) is that the binding capacity of the carboxylated beads in guanidine buffers is more reduced. When I was using SeraMag carboxylated beads to purify plasmid DNA according to the protocols described here (#5.1) I always got low amount of plasmid-DNA recovered compared to a control (like column method). The moment when I removed the guanidine buffer and used PEG buffers, the protocols started to work much better.
Now, the protocols.io link which you posted suggests that the beads are prepared in a PEG buffer, however, I think the guanidine salt causes some issues and this is why you have low recovery. Among things which I would try, would be to add more beads (make the stock solution more concentrated, like use 2 mL of beads /50 mL of stock solution when you prepare the beads), or don’t use SeraMag carboxylated beads, but try to adapt a protocol with silica coated beads from here. Silica works better in guanidine buffers, and it might solve your issue.
Best regards,
Sebastian
Hi guys,
Maybe you will find this question stupid, but I am curious if the magnetic bar used for stirring is in the NaOH solution, or in the water bath. I expect it to be in the NaOH solution (to mix it) but on the drawing it seems to be in the water bath which holds the round bottom flask (https://bomb.bio/wp-content/uploads/2018/09/1.1_BOMB_MNP_synthesis_V1.0.pdf ).
Thanks,
Sebastian
Hi guys,
We are not able to synthesize these beads as we don’t have the required anaerobic conditions. However, I was trying to optimize the protocol for the Sera-mag beads and it seems that for us, with our current protocol, it works well. I am even able to use it for midiprep. If anybody is interested, I am happy to share my protocol with Sera-mag beads.
Sebastian
Hello,
Sorry it was a confusion. I was referring to the printed rack for 8 tubes the “BOMB microtube rack”.
In the end I managed to fix it, by completely re-designing the rack from scratch. Sorry it wasn’t my intention to be rude or something, but some of the links which you have are not anymore available, and editing directly .stl files is not really straightforward, especially for me, who never designed anything in 3D software.
You have the link in your documentation to thingverse (https://www.thingiverse.com/thing:3199242 ) and in thingverse you have a link to tinkercad, however that link is not working. Yet the link from thingverse for the BOMB microplate (https://www.thingiverse.com/thing:3169529 ), has a valid link to the tinkercad design, and makes it very easy and nice to adjust the model if we want.
Anyway if somebody needs to modify the model to fit some specific magnets for themselves here is the link to tinkercad: https://www.tinkercad.com/things/38zjiHHNvhQ
The holes for the magnets are made to fit 15x10x3 mm magnets with 0.5 mm of the magnet sticking out from the hole to be as close as possible to the tube.
SebastianLater edit.
I noticed that on thingverse, there is a link to tinkercad to edit the same rack design. However, the link is not working anymore. If you have the model on tinkercad, could you please share it with us in order to edit it?
Thank you,
Sebastian
Hi Tim,
Plasmid extraction.
At least for the plasmid purification I don’t think the beads amount is an issue. I used the same amount of beads for both your #5.1 protocol and my current protocol. The only difference is that in my protocol I don’t have GuHcl in the N3 buffer, but instead is the N3 buffer from #5.2 (2.3 M KoAc). And for the binding (in my protocol) I make sure I have a 1:1 ratio between the supernatant recovered and the PEG buffer . I did both of these protocols in parallel and in the #5.1 I always see clumping (but the clumps can be broken is smaller pieces, however they can never be re-suspended in the PE wash buffer) as opposed to the one with PEG where I just vortex them and the beads form nicely a brownish solution with the PE wash buffer. Since I use the same amount of starting material for both protocols, the same culture, same plasmid, etc. I don’t think the issue is the amount of material. This is why my suspicion is towards the guanidine.
My experience with clumping is very vague, and everything relies on AmpureXP beads and preparation of libraries for nanopore sequencing. The only time I saw clumping was when I tried to use high molecular weight DNA to prepare a nanopore library as it was mentioned also here. http://seqanswers.com/forums/showthread.php?t=47522
Based on nanopore support, clumping is because the long strands of DNA bind tightly to the beads. Also because of this reason, eluting HMW DNA from carboxylated beads is hard, requires larger elution times and temperatures at around 65C (https://www.researchgate.net/post/How_can_I_fully_elute_whole_genomic_DNA_off_of_Sera-Mag_paramagnetic_beads ).
But this is not the case here, as the plasmid is 8kb or less.
Another reason why I don’t think that the bead amount is the issue is the following. I used the modified protocol by me for a Midiprep. In this case I used 70 mL of culture (exactly the same culture as above, this is why it is such an odd number) so roughly 14 times more than for a miniprep. But I increased the beads amount just 5 times. Yet the midiprep worked perfectly (785 ng/ul in 300 ul of elution buffer).
Somewhere, some people mention (no scientific citation for this) that the binding capacity of 1:50 diluted sera-mag beads is around 7 ug/ul of diluted beads. Yet I don’t have any citation for this.
GEL extraction:
I have a more concentrated beads solution for the Gel extraction 1:12.5. So 4 times more concentrated compared to what dilution is standard. I did not try to increase the volume because I think the isopropanol ratios are critical for the protocol. Either way the same result. At this stage honestly I don’t have time to look into this protocol and figure out why it does not work in my hand. The beads are visibly starting to clump the moment when I add to the dissolved agarose in GITC. I was thinking to add first the isopropanol, then to try to add the beads. I will see if I will have time to test this.
I think that sera-mag beads are not behaving exactly as your synthesized beads, and maybe this is why we get this issue with them.
Best regards,
Sebastian
Hi Tim,
I did exactly what you said, I diluted them 1:50, since many other protocols suggest the same thing. Also the washing was written on other protocols which I found on the internet.
Now I have some other questions specifically related to carboxyl beads. While the clean-up works perfectly and it is a good protocol, the other two protocols which I wanted to implement in our lab don’t work well with carboxyl coated beads.
The first one, the plasmid purification #5.1. For me it recovers kinda 1/3 of the amount compared to a commercial kit. I understand, your protocols are not optimized for carboxyl coated beads, so I am not surprised. However, I think there are several reason why this doesn’t work well.
1. The beads tend to clump in buffers which contain guanidine, and they don’t re-suspend in the wash buffer, they stay as small clumps, but never a uniform brown color as it should be when the beads are re-suspended. However, the beads can be re-suspended in the EB buffer, in the last step.
2. I think for the carboxyl beads the binding time should be larger. Also the volumes should be slightly lower to accommodate things in one microcentrifuge tubes. My experience (with ampure beads not specifically these ones, and for nanopore libraries) is that lower volumes and higher binding and elution times drastically increase the yield.
With these two things in mind I did a protocol for plasmid which uses a modified recipe for beads from the cleanup/size exclusion (so NaCl and PEG) and removed the guanidine. And I recover consistently around 80% of the plasmid compared to a commercial geneaid kit for plasmid. Eg. for a 5 mL LB culture I got 390 ng/ul (nanodrop) in 50 ul of EB compared to around 460 ng/ul with the geneaid kit. However, this is good enough for us. If you want I can share with you the protocols and test it with your synthesized beads and maybe publish it on the webpage for the people.
The other protocol which I tried to use is the gel purification. However, here I never managed to make the protocol work. First, using PEG does not help, if I actually use PEG and NaCl buffer, the beads form a sort of slime with something from the tube and they are really hard to work with.
With your original protocol my recovery is very low (speaking about large fragments above 600 bp) and I have a lot of salt contamination. No matter how much I wash (I tried 4 washes). Here I also notice that the beads form a clump, but this time around, I am literally unable to re-suspend the clump at all. They stick together no matter what. Again, my suspicion is the GITC in the buffer, but also somehow I think the agarose it is an issue, I think somehow the agarose can bind carboxylated beads (I know that in some nanopore protocols during the sequencing the pores die quickly and people think is a polysaccharide carryover, even if during the library prep you have 2-3 cleanups with ampure xp beads). Because of this experience with nanopore, I think that also agarose might bind to the beads and this is why I see this solid compact beads pellet. Interestingly, in some reactions during testing this did not occur, and I have no idea what was the case there.
Did you guys observe these issues in gel purification during testing of the gel extraction?
Thank you,
Sebastian
