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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!
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Ampure has a protocol for 96 and 384 well plates. Link to protocol (PDF):
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For tighter sizes pippin or Caliper labchip XT although no plate format for those..Comment
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thanks. but that's not size selection like cutting a narrow band from the gel, but rather just DNA purification.Originally posted by Zaag View PostComment
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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.Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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