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Old 09-09-2016, 04:42 PM   #21
nucacidhunter
Jafar Jabbari
 
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Binding DNA to beads is one sided but size selection can be left-sided, right-sided or double-sided. Following link has good explanation and discussion on the mechanism.

http://core-genomics.blogspot.com.au...eads-work.html

There are different brands of beads sold for clean up or size selection but their cut off and recovery efficiency varies, so you would need to do some trial to find the optimum bead/DNA ratio for your application.

If you spike large fragment library into short fragment one you probably will get small number of sequence reads from large fragments.

0.55x will cut somewhere around 600 bp depending on bead brand.


Edit: Ligation reactions containing PEG will affect size cut off.

Last edited by nucacidhunter; 09-09-2016 at 11:20 PM.
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Old 09-13-2016, 02:14 AM   #22
Lovro
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Thank you for the link.

I will use the beads that come with Nextera kits:



Are there any specificities on them I should know about ?

In regard to the edit: More peg means shorter fragments will be included ? Do you happen to know, if the effect will be large with Illumina TruSeq PCR-free kit?


Thank you!
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Old 09-13-2016, 03:04 AM   #23
nucacidhunter
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The beads in the picture are from TruSeq kit. They are SPRIselect or AMPure XP from Beckman. Nextera kits do not contain any beads although Nextera XT comes with library normalization beads.

TruSeq PCR free ligation reaction does not seem to have high concentration of PEG so the effect will be minimal and anyway there is a second clean up as well.
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Old 09-13-2016, 05:15 AM   #24
Lovro
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They are from Nextera exome kit, not generic nextera.
http://www.illumina.com/products/nex...xome-kits.html

tnx for all the info!
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