Shearing digested DNA for RADseq

IanAWarren · 11-16-2016, 06:14 AM

Hello,

I am making libraries for RADseq. I am trying out the Covaris ME220 for shearing the digested DNA. The shearing seems to be highly inefficient compared to what is given in the protocol book (using whole genomic DNA). Does anyone have any advice about ths in general?

I have identified three possible problems:

1) I am starting with digested DNA and I read somewhere that it does not shear as efficiently. The average size of my DNA should be 4096bp and I need to get it to ~500bp

2) Since I am going from the digestion to the ligation and then the shearing directly I still have the old enzymes, buffer, and some rATPs in the mixture.

3) I might be working with a low concentration of DNA, but I do not think this is right. I have about 1ug in 130ul, which is not low concentration to me.

I am going to perform some trial and error and hopefully find a solution, but my initial efforts have proved ineffective. I think i was being too tentative.

Thank you in advance for any help that you can provide

huguesparri · 11-16-2016, 08:33 AM

Hello,

Usually, we perform a AMPure XP purification step after the P1 ligation and we do not face any issue with the sonication. Maybe you should give it a try...

H.

kerplunk412 · 11-16-2016, 01:00 PM

I have Covaris digested DNA of 1-2 kb and it works well. I think your problem is #2.

IanAWarren · 11-17-2016, 01:15 AM

Hello,

Thank you for your answers.

What conditions did you use for your Covaris machine? We have not got close to the sizes we want (actually too small now) and we are fine tuning. We have just found that the conditions required are very different from what is given in the protocol book.

I will suggest the purification too.

huguesparri · 11-17-2016, 05:19 AM

Usually we use a Bioruptor because it's located in our building.
But we also have access to a S220 in the university. When we used it, the parameters were as follow:
Volume: 130µl.
DNA: 1 µg.
sonication time: 60 sec.
Duty Cycle : 10% , Intensity (Peak Incident Power), 5 (175), Cycles/Burst 200.

Regards,

H.

PS:
An answer from the IGF of Montpellier to the IGF of Lyon ^^

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11-16-2016, 06:14 AM	#1
IanAWarren Member Location: Cambridge Join Date: Feb 2013 Posts: 11	Shearing digested DNA for RADseq Hello, I am making libraries for RADseq. I am trying out the Covaris ME220 for shearing the digested DNA. The shearing seems to be highly inefficient compared to what is given in the protocol book (using whole genomic DNA). Does anyone have any advice about ths in general? I have identified three possible problems: 1) I am starting with digested DNA and I read somewhere that it does not shear as efficiently. The average size of my DNA should be 4096bp and I need to get it to ~500bp 2) Since I am going from the digestion to the ligation and then the shearing directly I still have the old enzymes, buffer, and some rATPs in the mixture. 3) I might be working with a low concentration of DNA, but I do not think this is right. I have about 1ug in 130ul, which is not low concentration to me. I am going to perform some trial and error and hopefully find a solution, but my initial efforts have proved ineffective. I think i was being too tentative. Thank you in advance for any help that you can provide

11-16-2016, 08:33 AM	#2
huguesparri Member Location: Montpellier (France) Join Date: May 2008 Posts: 93	Hello, Usually, we perform a AMPure XP purification step after the P1 ligation and we do not face any issue with the sonication. Maybe you should give it a try... H.

11-16-2016, 01:00 PM	#3
kerplunk412 Senior Member Location: Bioo Scientific, Austin, TX, USA Join Date: Jun 2012 Posts: 119	I have Covaris digested DNA of 1-2 kb and it works well. I think your problem is #2.

11-17-2016, 01:15 AM	#4
IanAWarren Member Location: Cambridge Join Date: Feb 2013 Posts: 11	Hello, Thank you for your answers. What conditions did you use for your Covaris machine? We have not got close to the sizes we want (actually too small now) and we are fine tuning. We have just found that the conditions required are very different from what is given in the protocol book. I will suggest the purification too.

11-17-2016, 05:19 AM	#5
huguesparri Member Location: Montpellier (France) Join Date: May 2008 Posts: 93	Usually we use a Bioruptor because it's located in our building. But we also have access to a S220 in the university. When we used it, the parameters were as follow: Volume: 130µl. DNA: 1 µg. sonication time: 60 sec. Duty Cycle : 10% , Intensity (Peak Incident Power), 5 (175), Cycles/Burst 200. Regards, H. PS: An answer from the IGF of Montpellier to the IGF of Lyon ^^