DNA size range for Covaris sonicator?

glassfish · 07-20-2011, 08:18 AM

I am doing a Raindance project right now. I tried to concatenate the PCR products before fragmentation　by Covaris. But the best I can get so far is between 1500bp-2000bp. I was told that PCR products should　ligate to more than 5kb, so that Covaris can work well to shear them to 200bp.

I am wondering whether Covaris will still work well with DNA size range 1.5-2kb? Since I really don't know how to improve the ligation...

Thank you very much!

pmiguel · 07-20-2011, 12:29 PM

Did you blunt end and phosphorylate your PCR products prior to ligation?

Many polymerases (eg, Taq polymerase) add a non-templated 3' A. And PCR oligos are not 5' phosphorylated unless you specify that they should be when ordering them.

--
Phillip

glassfish · 07-20-2011, 12:45 PM

Thanks for you reply Philip! I used end-repair kit before ligation, it blunts the end and phosphorylates 5'. The peak at Bioanalyzer after ligation is about 2kb. I am wondering how people could get it to 4kb or above...

Quote:

Originally Posted by pmiguel

Did you blunt end and phosphorylate your PCR products prior to ligation?

Many polymerases (eg, Taq polymerase) add a non-templated 3' A. And PCR oligos are not 5' phosphorylated unless you specify that they should be when ordering them.

--
Phillip

pmiguel · 07-20-2011, 02:20 PM

Run the ligation longer?

I don't think the sonicator has any difficulty shearing 2kb stuff. Is the issue you are trying to avoid an inability to fragment short products, or excess terminal reads from the ends of the PCR products?

--
Phillip

Hamid · 07-22-2011, 01:09 PM

Hi Glassfish,

You can certainly shear 1.5kb-2kb fragments down to 200bp using your Covaris instruments. Quite a few people currently do that with amplicons. All you will have to do is to use the same settings for generating 200bp ( 10%dc/5i/200cpb/180 seconds) in 130ul volume in our microTUBEs and carry out a time course of up to 1 minute beyond the 180 seconds.

Thank you

Hamid

pmiguel · 07-23-2011, 08:51 AM

Yes, that was my impression. Nebulization has trouble fragmenting when starting with short fragments but not really an issue with a sonicator.

That said, there is a second issue unrelated to fragmentation method. You can get strong PCR product end bias in your final sequence if some care is not taken. Ligation is one way of mitigating that. But if something about your PCR product ends is making them hard to ligate even after end polishing, end bias may not be an issue for you.

--
Phillip

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07-20-2011, 08:18 AM	#1
glassfish Junior Member Location: US Join Date: Jun 2011 Posts: 6	DNA size range for Covaris sonicator? I am doing a Raindance project right now. I tried to concatenate the PCR products before fragmentation　by Covaris. But the best I can get so far is between 1500bp-2000bp. I was told that PCR products should　ligate to more than 5kb, so that Covaris can work well to shear them to 200bp. I am wondering whether Covaris will still work well with DNA size range 1.5-2kb? Since I really don't know how to improve the ligation... Thank you very much!

07-20-2011, 12:29 PM	#2
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Did you blunt end and phosphorylate your PCR products prior to ligation? Many polymerases (eg, Taq polymerase) add a non-templated 3' A. And PCR oligos are not 5' phosphorylated unless you specify that they should be when ordering them. -- Phillip

07-20-2011, 02:20 PM	#4
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Run the ligation longer? I don't think the sonicator has any difficulty shearing 2kb stuff. Is the issue you are trying to avoid an inability to fragment short products, or excess terminal reads from the ends of the PCR products? -- Phillip

07-22-2011, 01:09 PM	#5
Hamid Senior Member Location: San Diego, CA Join Date: Sep 2009 Posts: 105	Hi Glassfish, You can certainly shear 1.5kb-2kb fragments down to 200bp using your Covaris instruments. Quite a few people currently do that with amplicons. All you will have to do is to use the same settings for generating 200bp ( 10%dc/5i/200cpb/180 seconds) in 130ul volume in our microTUBEs and carry out a time course of up to 1 minute beyond the 180 seconds. Thank you Hamid

07-23-2011, 08:51 AM	#6
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Yes, that was my impression. Nebulization has trouble fragmenting when starting with short fragments but not really an issue with a sonicator. That said, there is a second issue unrelated to fragmentation method. You can get strong PCR product end bias in your final sequence if some care is not taken. Ligation is one way of mitigating that. But if something about your PCR product ends is making them hard to ligate even after end polishing, end bias may not be an issue for you. -- Phillip