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Old 12-22-2016, 08:40 AM   #1
mjg
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Location: Argentina

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Post SPRI dilution in Illumina Library Prep

Hello everyone,
I'm actually a newbie in this field, and this forum has helped me a lot with some issues.
I've been reading a few Illumina protocols, like TruSeq nano or TruSeq DNA PCR-Free, and in both protocolos there is a dual size selection.
I want to know why there is a bead dilution step before the large fragment removal?. For example,
109.25 ul beads + 74.75 PCR grade water = 184 ul final volume
Then you must use 160 ul of this mix with your sample (equivalent to 95 ul beads)

Is not simplier to add this equivalent bead volume to your sample, regarding to get the same finale volume? (in this case 260 ul)

I hope I've been clear enough.

Thanks!
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Old 12-22-2016, 05:47 PM   #2
nucacidhunter
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This approach is not necessary and it can be done without diluting beads and using different bead volumes to keep the bead to sample volume ratios similar. Hovewer, dilution helps to achieve consistent results by buffering inaccuracy in pipetting small volumes. Other reason might be that Illumina keeps the protocols for different size selections similar by using different dilutions but the same volume of diluted beads for 350 and 550bp insert libraries. Higher volumes also can help consistency in high throughput protocols that relies on shaking to mix beads and reactions.

Last edited by nucacidhunter; 12-22-2016 at 05:50 PM.
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Old 12-23-2016, 03:26 AM   #3
mjg
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Quote:
Originally Posted by nucacidhunter View Post
This approach is not necessary and it can be done without diluting beads and using different bead volumes to keep the bead to sample volume ratios similar. Hovewer, dilution helps to achieve consistent results by buffering inaccuracy in pipetting small volumes. Other reason might be that Illumina keeps the protocols for different size selections similar by using different dilutions but the same volume of diluted beads for 350 and 550bp insert libraries. Higher volumes also can help consistency in high throughput protocols that relies on shaking to mix beads and reactions.
Thanks nucacidhunter,
you helped me a lot.
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