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