Kun:LabNotes/ASE/2007-11-5
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. I didn't have enough BciVI, so I added only 1~2ul enzyme to Lib #2. It turned out that I got complete digestion in all reactions, indicating I don't need 4ul of enzyme.
File:2007-11-05-BciVI-cut.jpg
65C 10min to inactivate BciVII.
Lambda exo digestion Purify with Qiaquick column, eluted in 50ul water. Add 5ul 10x lambda exo buffer, 2ul lambda exo, 37C 2h, 75C 10min.
Add 4ul USER enzyme, 37C 2h, check with 6% TBU gel.