high throughput DNA size selection?

polyhedron · 11-04-2010, 11:02 PM

Hi! I'm asking is there any method out for a large quantity of DNA size selection, something like with 96-well plate. The traditional way is to excise agaroase gel and do DNA purification, but it's too labor-intensive for hundreds of samples. I think it would be perfect to do size selection in a 96-well plate. But something like Sephadex seems not able to do such selection (e.g. 250-300bp). Any idea? Thanks!

Zaag · 11-05-2010, 01:52 AM

Ampure has a protocol for 96 and 384 well plates. Link to protocol (PDF):

http://www.beckmangenomics.com/docum...000387v001.pdf

josdegraaf · 11-05-2010, 07:41 AM

For tighter sizes pippin or Caliper labchip XT although no plate format for those..

polyhedron · 11-08-2010, 08:10 AM

Quote:

Originally Posted by Zaag

Ampure has a protocol for 96 and 384 well plates. Link to protocol (PDF):

http://www.beckmangenomics.com/docum...000387v001.pdf

thanks. but that's not size selection like cutting a narrow band from the gel, but rather just DNA purification.

pbluescript · 11-08-2010, 08:41 AM

You can use the ratio of AMPure beads to sample to select a specific size range without using a gel. Since the beads are in a PEG/salt solution, altering the ratio of beads/sample will solubilize different sizes of DNA fragments. For example, using 0.6X AMPure beads removes DNA <500bp and using 1.2X removes DNA <150bp. Using two different ratios of beads will allow you to select a certain size range of your library. Using a 0.6X ratio will cause binding of DNA fragments >500bp, but the smaller fragments will still remain in the supernatant. You can take that supernatant and add it to a higher concentration of AMPure beads and then take the DNA bound to the beads to get a size selection based on the ratios you used. With the 0.6X and 1.2X ratios, your library would range from 150-500bp. Altering the ratios by 0.1X steps will allow you to collect narrower size ranges. This type of size selection isn't extremely accurate so you should only use it if appropriate for your application. Be sure to test this out on some 100bp ladder or something similar since there is a little bit of lot-to-lot variation.

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11-04-2010, 11:02 PM	#1
polyhedron Member Location: China Join Date: Aug 2009 Posts: 12	high throughput DNA size selection? Hi! I'm asking is there any method out for a large quantity of DNA size selection, something like with 96-well plate. The traditional way is to excise agaroase gel and do DNA purification, but it's too labor-intensive for hundreds of samples. I think it would be perfect to do size selection in a 96-well plate. But something like Sephadex seems not able to do such selection (e.g. 250-300bp). Any idea? Thanks!

11-05-2010, 01:52 AM	#2
Zaag Senior Member Location: Amsterdam Join Date: Nov 2009 Posts: 112	Ampure has a protocol for 96 and 384 well plates. Link to protocol (PDF): http://www.beckmangenomics.com/docum...000387v001.pdf

11-05-2010, 07:41 AM	#3
josdegraaf Member Location: Germany Join Date: Mar 2010 Posts: 33	For tighter sizes pippin or Caliper labchip XT although no plate format for those..

11-08-2010, 08:41 AM	#5
pbluescript Senior Member Location: Boston Join Date: Nov 2009 Posts: 224	You can use the ratio of AMPure beads to sample to select a specific size range without using a gel. Since the beads are in a PEG/salt solution, altering the ratio of beads/sample will solubilize different sizes of DNA fragments. For example, using 0.6X AMPure beads removes DNA <500bp and using 1.2X removes DNA <150bp. Using two different ratios of beads will allow you to select a certain size range of your library. Using a 0.6X ratio will cause binding of DNA fragments >500bp, but the smaller fragments will still remain in the supernatant. You can take that supernatant and add it to a higher concentration of AMPure beads and then take the DNA bound to the beads to get a size selection based on the ratios you used. With the 0.6X and 1.2X ratios, your library would range from 150-500bp. Altering the ratios by 0.1X steps will allow you to collect narrower size ranges. This type of size selection isn't extremely accurate so you should only use it if appropriate for your application. Be sure to test this out on some 100bp ladder or something similar since there is a little bit of lot-to-lot variation.