[PStepper]

Hi everyone,

this is the main discussion space for BOMB protocol #9.1 Bisulfite conversion.
Here you can post all your questions and feedback!

[PStepper]

reserved space

[Michael Dunnet]

Hey,

Just a quick question regarding the conversion step of the protocol. I’ve been getting lower conversion efficiencies compared to the previous kit I had been using (the EZ DNA methylation MagPrep kit by Zymo). I’ve noticed that the temperatures in the conversion step are much lower than in the Zymo kit despite everything else been very similar. I’m curious about the reasoning for this and whether or not you think I could improve the efficiency by increasing these temperatures.

Cheers,

Michael

[PStepper]

Hi Michael,

As you probably know it’s important to find the right balance between cytosine conversion, protection of methylated cytosines, and keeping DNA fragmentation to a minimum. But if your conversion efficiencies are lower, you’re right that I’d expect that either increasing the conversion temperature or incubation time (or even both) would boost the conversion rate. Maybe just give it a try with the 64 °C incubation for your application and report back the results to the community? And I’d advise to keep an eye out for DNA integrity and 5mC conversion (if you want to test this).

Maybe also have a look at these papers:

https://academic.oup.com/nar/article/36/22/e150/1195141#20603058

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0093933

Best

Peter

[Jiyi]

hi everyone,

Is your recovery rate higher than 50%? My recovery is only 10% when I followed the protocol. Only difference is the silica-coated magnetic beads. I purchased the beads from other company. Its concentration is 50mg/ul and the size is 100nm.

The recovery of the EZ DNA methylation MagPrep kit(Zymo) is about 50-70%.

Best,

Ji

[Michael Dunnet]

Hi Jiyi,

I seem to get equivalent recovery rates with both BOMB and the EZ DNA methylation MagPrep kit. One thing that might be causing the issue is the washing steps. I know the zymo kit asks you to re-suspend the beads upon each wash; however, when I use the BOMB kit I keep the beads on the magnet and just add the ethanol to the tube for around 30s before removing it.

If that is something you already do I’m unsure on what the issue could be. Nevertheless, I hope this is helpful.

 

Cheers,

Michael

[Jiyi]

Hi Michael,

Thanks for your reply, I repeat my experiment several time as keeping  beads on the magnet at the wash steps. The recovery raised to 20%, which is still low to me.

I am wondering that maybe the key problem is my beads.  I will update my progress if there are some good news.

Best,

Ji

 

[Jeremy Abels]

Hello,

I have a question regarding the beads used for this protocol. Is it possible to use carboxyl coated beads in lieu of silica coated beads. If so, what buffer is appropriate to use as the binding buffer?

Thank you,

-Jeremy

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