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Old 09-29-2014, 12:12 PM   #3
AdrianaGeldart
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Location: Boston, MA

Join Date: Feb 2014
Posts: 6
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Dear yxl,

I am a member of the Kapa Scientific Support & Applications Team and would be happy to address your question.

To perform the first size cut (to exclude fragments corresponding to library inserts larger than ~450 bp), you would add 30 uL of AMPure XP Reagent to 50 uL of DNA. This is the 0.6X cut as you correctly described.

The second step would be to transfer the supernatant containing DNA fragments smaller than ~450 bp to a new plate and discard the plate with the beads to which DNA fragments larger than ~450 bp are bound. The second size cut is then performed (to retain fragments corresponding to library inserts larger than ~250 bp) by adding 0.2 volumes of Agencourt AMPure XP reagent to the first cut supernatant. This would be adding 10 uL of reagent to 75 uL of DNA. This 0.2 volumes corresponds to the volume of DNA prior to the first cut (i.e. 50 uL). We have found 0.2 volumes to be optimal. If you decrease the difference between the two cuts to <0.2, the peak width decreases, but at the cost of a higher loss of DNA. If you increase the difference between the two cuts to >0.2, recovery is better, but the peak width tends to broaden.

I hope this helps to clarify the size selection. If you would like further explanation, please feel free to call the Support Line at 1-855-KAPA-BIO or email us at support@kapabiosystems.com.

Best Regards,
Kapa Biosystems Support
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