Kun:LabNotes/ASE/2007-11-5
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Exp. 1
Purify PCR amplicons with QiaQuick columns, 400ul reactions per column. During the purification, I wasn't completely sure I added the amplicons to the tubes with correct label. There is a possibility that I did it in the wrong order:
Lib #1 -> tube #2 43ng/ul x ~100ul Lib #2 -> tube #3 63ng/ul x ~100ul Lib #3 -> tube #4 53ng/ul x ~100ul Lib #4 -> tube #5 54ng/ul x ~100ul Lib #5 -> tube #1 41ng/ul x ~100ul Lib #1 -> tube #1.2 86ng/ul x ~50ul
I decided to proceed with the purification. I purified another 400ul Lib #1 with a separate column.
I also performed ethanol precipitation on 400ul of Lib #2-5. In addition, I repeat PCR on Lib #1&2 using iTaq.
BciV digestion
To the ~45ul of column purified tube #1.2 and ~45ul ethanol precipitated Lib #2-5, add 5ul NEBuffer 4, 4ul BciVI, 37C 3h, keep @4C, check with 8% TBE gel.
File:2007-11-05-BciVI-cut.jpg