Magnesium + heat = Shear DNA?

herter · 01-31-2017, 04:00 PM

I know in some RNA-Seq protocols magnesium and heat are used to shear the RNA. I'm curious if magnesium and heat will also shear DNA?
Thanks!

ECO · 01-31-2017, 07:22 PM

Without a 2' -OH, the DNA won't shear.

Magnesium is in high concentrations in every PCR buffer also.

nucacidhunter · 01-31-2017, 09:02 PM

If you are looking for metal ion based DNA shearing see the following:

https://beng.soe.ucsc.edu/sites/defa...esis_Final.pdf

ECO · 02-01-2017, 10:41 AM

Anyone tried this? ^^

Seems gloriously too good to be true.

herter · 02-01-2017, 11:01 AM

Thanks for the replies! Any recommendations on chemical or enzymatic shearing for DNA and RNA (TNA) at the same time? We do mechanical shearing now for TNA, but hoping to find a faster/easier approach.

nucacidhunter · 02-01-2017, 12:53 PM

It is possible to use combination of DNA fragmenting enzyme such as NEB fragmentase with RNase III. The issue would be buffer and incubation temperature and time requirement which can be overcome by treating with one enzyme followed by dilution and adding other enzyme and buffer without a need for clean up in between and optimising enzyme quantity.

Adobe Freeman · 01-12-2020, 06:20 AM

recently I have tried it
Add 400ng Lambda DNA(48.5K) into 1X Taq Premix with 2mM MgCl2,
it shows clearly fragmentation more than 1min at 95C if more 4min median of fragment is around several Kilo; 95C 16min most of fragment is small than 1K.

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01-31-2017, 04:00 PM	#1
herter Junior Member Location: Saint Louis Join Date: Feb 2015 Posts: 8	Magnesium + heat = Shear DNA? I know in some RNA-Seq protocols magnesium and heat are used to shear the RNA. I'm curious if magnesium and heat will also shear DNA? Thanks!

01-12-2020, 06:20 AM	#7
Adobe Freeman Junior Member Location: HK Join Date: Jun 2019 Posts: 1	DNA Fragmentation at Taq Premix recently I have tried it Add 400ng Lambda DNA(48.5K) into 1X Taq Premix with 2mM MgCl2, it shows clearly fragmentation more than 1min at 95C if more 4min median of fragment is around several Kilo; 95C 16min most of fragment is small than 1K.

01-31-2017, 07:22 PM	#2
ECO --Site Admin-- Location: SF Bay Area, CA, USA Join Date: Oct 2007 Posts: 1,358	Without a 2' -OH, the DNA won't shear. Magnesium is in high concentrations in every PCR buffer also.

01-31-2017, 09:02 PM	#3
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	If you are looking for metal ion based DNA shearing see the following: https://beng.soe.ucsc.edu/sites/defa...esis_Final.pdf

02-01-2017, 10:41 AM	#4
ECO --Site Admin-- Location: SF Bay Area, CA, USA Join Date: Oct 2007 Posts: 1,358	Anyone tried this? ^^ Seems gloriously too good to be true.

02-01-2017, 11:01 AM	#5
herter Junior Member Location: Saint Louis Join Date: Feb 2015 Posts: 8	Thanks for the replies! Any recommendations on chemical or enzymatic shearing for DNA and RNA (TNA) at the same time? We do mechanical shearing now for TNA, but hoping to find a faster/easier approach.

02-01-2017, 12:53 PM	#6
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	It is possible to use combination of DNA fragmenting enzyme such as NEB fragmentase with RNase III. The issue would be buffer and incubation temperature and time requirement which can be overcome by treating with one enzyme followed by dilution and adding other enzyme and buffer without a need for clean up in between and optimising enzyme quantity.