E-gel vs BluePippin for narrow size selection???

Sudharsan · 10-18-2016, 07:29 AM

Hi Everyone,

I am facing a problem with size selection of Covaris fragmented DNA. I would require insert sizes in the narrow range 700-800 bp for library preparation. BluePippin was not able to provide a narrow size range for the 750 bp library.
So I was wondering if I can use the E-gel 2% size select for this narrow size selection.
Have you ever tried getting narrow sizes using E-gel size select for the sizes 700-800 bp? What is the recovery rate of the library following size selection? What is your opinion on use of E-gel for size selection compared to BluePippin?

It will be very helpful for me if you can share some details.
Looking forward for your reply. Thank you very much.

Regards,
Sudharsan

sward · 10-21-2016, 03:50 AM

Hello,

I would say although gel size selection is a lot more hands on, you definitely get a narrower peak if done correctly. I've only ever tried for 500bp but that worked a lot better using a gel rather than pippin - i just couldn't get the parameters right with the pippin. Also with pippin, in my experience, I have had to load all of my sample and once its done the size selection that's it - its gone, its very hard to recover anything if it doesn't size select correctly. Whereas with gel at least you can take multiple cuts (target, lower, upper, lower lower and upper upper) and freeze them in case your target slice isn't as close to 750bp as you want it. Also - definitely recommend testing your gel/ladder with some controls as I found I would actually have to cut at the 400bp ladder mark in order to get my sample at 500bp.

hope this helps a little bit

thermophile · 10-21-2016, 06:37 AM

you could also try double bead cleaning. Look at p. 61 https://www.broadinstitute.org/files...PrepSlides.pdf

nucacidhunter · 10-21-2016, 03:38 PM

I think Pippin will give you the tightest cut in comparison to other methods if correct cassette and loading quantity was used. If you try to get very tight cut then you have to start with a lot more DNA as only a small fraction of DNA will be recovered.

E-gel would be similar (not better) to Pippin but controlling size will be difficult. Manual gel will result in significantly higher number of smaller shadow fragments.

Sudharsan · 10-26-2016, 01:32 AM

Thank you everyone for your suggestions. As of now BluePippin is not able to provide the narrowest cut needed. So I will try out bead based double selection and also E-gel and let you know how it works.

kerplunk412 · 10-27-2016, 07:51 AM

In my experience the yield from the E-gel is terrible, which makes sense as it doesn't take DNA very long to cross the buffer well. The Pippin should be able to give you as good or better resolution as the E-gel, but with better yield. You may need to optimize the collection range though; for the purpose I was using it for, recovering a 150 bp band with high resolution, the optimal collection range was fairly different from what one would expect.

That being said, I also think that a classic gel purification may be your best bet, for the reasons that sward mentioned.

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10-18-2016, 07:29 AM	#1
Sudharsan Junior Member Location: Germany Join Date: Sep 2016 Posts: 2	E-gel vs BluePippin for narrow size selection??? Hi Everyone, I am facing a problem with size selection of Covaris fragmented DNA. I would require insert sizes in the narrow range 700-800 bp for library preparation. BluePippin was not able to provide a narrow size range for the 750 bp library. So I was wondering if I can use the E-gel 2% size select for this narrow size selection. Have you ever tried getting narrow sizes using E-gel size select for the sizes 700-800 bp? What is the recovery rate of the library following size selection? What is your opinion on use of E-gel for size selection compared to BluePippin? It will be very helpful for me if you can share some details. Looking forward for your reply. Thank you very much. Regards, Sudharsan

10-21-2016, 03:50 AM	#2
sward Junior Member Location: United Kingdom Join Date: Apr 2016 Posts: 3	Hello, I would say although gel size selection is a lot more hands on, you definitely get a narrower peak if done correctly. I've only ever tried for 500bp but that worked a lot better using a gel rather than pippin - i just couldn't get the parameters right with the pippin. Also with pippin, in my experience, I have had to load all of my sample and once its done the size selection that's it - its gone, its very hard to recover anything if it doesn't size select correctly. Whereas with gel at least you can take multiple cuts (target, lower, upper, lower lower and upper upper) and freeze them in case your target slice isn't as close to 750bp as you want it. Also - definitely recommend testing your gel/ladder with some controls as I found I would actually have to cut at the 400bp ladder mark in order to get my sample at 500bp. hope this helps a little bit Last edited by sward; 10-21-2016 at 04:23 AM.

10-21-2016, 06:37 AM	#3
thermophile Senior Member Location: CT Join Date: Apr 2015 Posts: 243	you could also try double bead cleaning. Look at p. 61 https://www.broadinstitute.org/files...PrepSlides.pdf __________________ Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.

10-21-2016, 03:38 PM	#4
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	I think Pippin will give you the tightest cut in comparison to other methods if correct cassette and loading quantity was used. If you try to get very tight cut then you have to start with a lot more DNA as only a small fraction of DNA will be recovered. E-gel would be similar (not better) to Pippin but controlling size will be difficult. Manual gel will result in significantly higher number of smaller shadow fragments.

10-26-2016, 01:32 AM	#5
Sudharsan Junior Member Location: Germany Join Date: Sep 2016 Posts: 2	Thank you everyone for your suggestions. As of now BluePippin is not able to provide the narrowest cut needed. So I will try out bead based double selection and also E-gel and let you know how it works.

10-27-2016, 07:51 AM	#6
kerplunk412 Senior Member Location: Bioo Scientific, Austin, TX, USA Join Date: Jun 2012 Posts: 119	In my experience the yield from the E-gel is terrible, which makes sense as it doesn't take DNA very long to cross the buffer well. The Pippin should be able to give you as good or better resolution as the E-gel, but with better yield. You may need to optimize the collection range though; for the purpose I was using it for, recovering a 150 bp band with high resolution, the optimal collection range was fairly different from what one would expect. That being said, I also think that a classic gel purification may be your best bet, for the reasons that sward mentioned.