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Old 08-12-2015, 02:05 PM   #3
Lucigen_NGS_Service
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Location: Madison, WI

Join Date: Aug 2015
Posts: 2
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Hello Dr. Miranda and thank you for your inquiry. In our follow-up with you, you noted the primary DNA loss occurred after the adaptor ligation (instead of before as stated in your initial post), which is atypical for users of either the long mate pair or ion torrent kits. While some loss during the cleanup of the adaptor ligation is normal and expected, yours is the first report of losses to that high an extent. We identified that it may of help to check your shearing on g-TUBES (Covaris) prior to ligating adaptors, as we have noted that these devices perform as expected provided you normalize masses to those in the manufacturer’s table, but the mass you chose of 11.5 mcg is between the 8 and 15 mcg settings. In cases that fall outside, it is best to test the settings used to see if all liquid passes through the device, and once done, what the sheared DNA looks like on a gel. We have encountered higher DNA loss in the adaptor ligation cleanup when DNA shearing was incomplete due to sample volume not completely passing through the device. We welcome any follow-up discussion or questions in either this forum or we can always be reached at techserv@lucigen.com.
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