- This topic has 21 replies, 11 voices, and was last updated July 11, 2021 at 7:19 pm by .
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Hi everyone,
this is the main discussion space for BOMB protocol #5.1 plasmid DNA extraction from E. coli.
Here you can post all your questions and feedback!We actually never tried plasmid preps with the carboxyl-coated beads. We optimized them with the silica-beads quite a while before we even synthezised the first batch of carboxyl-beads, so there was no need for that. I don’t see why it wouldn’t work with them, but I can’t give you a guaranty as the buffers were optimized for the silica-beads. I would just give it a try with a few Minipreps and see how it works out. If you do them in Eppendorf vials, it takes about as long as a Miniprep with the silica coloumns.
It seems that the Nanodrop over-estimates the DNA concentration of our plasmid minipreps when we use our silica-coated beads. Does anyone have a suggestion? Many thanks
How does the uv/vis spectrum looks like? Do you have shouldering towards 230? We got rid of this problem when we washed once more with EtOH (we did not wash enough and there was residual GuHCl). Potentially it can also be contamination with RNA. Make sure you use fresh (or at least checked) RNase in the buffer. You can check this by running an agarose gel and stain with EtBr, if there is small molecule (~100 bp) band, it would suggest you still have RNA in the sample.
Hope this helps,
Tomek
Hi,
Thanks for the provided protocols, I appreciate the effort ! I made my own beads and succesfully preped the first mini preps. I did use the buffers I had left over from a ROCHE kit since they are nearly the same.
I have a few questions:
In step 8 you state to mix first with ethanol for 5 minutes and than add the beads. I did add ethanol and the beads together, and than mixed in a top over top rotor for 15 minutes (I work with 1.6 ml Eppendorf tubes). Is that ok ?
I noticed that after elution from the beads quite some volume is “taken up” by the beads: I used 75 µl Eb and got back 50 µl (I used 40 µl beads). This is as expected ?
Thanks,
Cor
Hey Cor,
Mixing the beads with the ethanol before adding the supernatant is perfectly fine. In this step it’s only critical to have the ethanol in there, as it drastically increases your yield.
Yes, it’s expected that the beads will take up some volume, especially if they were dried thoroughly.
I hope you were able to isolate some good yields and sorry for the delayed answer.
Cheers
PhilHey Bomb Beads Team!
First and foremost, thanks for this amazing resource!
Second and secondmost. Quick question. Would this and/or 5.2 protocol work with sermag beads? I gave it a shot and did not get very good results. But I just now realized that you say it was optimized for the silica beads and am wondering if it won’t work with other beads.
Also, I was wondering why you use ethanol in your binding step? I’m used to using beads for DNA size selections and was under the impression based on that protocol that using so much ethanol would inhibit binding?
Thanks!
SarahHi Sarah,
Sorry for the late response! We’re just moving our lab so there’s quite some work to do away from the laptop ^^
Firstly, thanks a lot! We’re glad you like the protocols 🙂
To your first question: The Sera-Mag Beads are built a little bit differently than our beads. While Sera-Mag have a polystyrene core coated with one or even two magnetic layers (here) ours have a ferrite core which is directly coated with either silica- or carboxyl. Therefore, they’ll differ quite bit in size and will most likely exhibit a different behaviour regarding binding and settling under the conditions in the protocol. Although, I personally never tried the Sera-Mag Beads with the #5.2 protocol, we used them in others. E.g. in #6.3 our collaborators in NZ compared them in TNA isolation from tissues, with a lower yield (as you described it). I will ask them about the exact procedure (our protocol with Sera-Mag Beads, or the complete Sera-Mag Kit) and come back to you on this.
To your second question: Generally, we have two states of the nucleic acid in our protocols: in solution or binding to the beads. The latter happens via chaotropic bridges formed by the GITC or Gu-HCl e.g. between the DNA and the silica-surface. This state is facilitated when the DNA has a harder time staying in solution, where the ethanol comes into play. It has a far lower dielectric constant than water, which means is can shield positive and negative charges in the solution much poorer than water. As the negatively charged DNA is attracted to the positive salt ions, ethanol has a much harder time keeping them apart (and avoid precipitation) than water has. You will also find, that basically every protocol for nucleic acid isolation and purification using either silica-beads or -columns use ethanol in their washing buffer.
I hope this helps you with your questions 🙂 I will come back to you on the tissue protocol.
Cheers
Hi Sarah/Phil – just a quick message to confirm that for the TNA from tissues purification using Sera-Mag or BOMB beads; exactly the same protocol was used. As Phil mentioned, there was slightly higher yield for most tissues using BOMB beads…
Cheers,
Tim
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
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