Kun:LabNotes/ASE/2007-11-5
Exp. 1[edit]
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.
Lib #1 61ng/ul x ~50ul Lib #2 88ng/ul x ~50ul Lib #3 34ng/ul x ~50ul Lib #4 58ng/ul x ~50ul Lib #5 44ng/ul x ~50ul
Add 5ul 10x lambda exo buffer, 2ul lambda exo, 37C 2h, 75C 10min.
Add 4ul USER enzyme, 37C 2h, check with 6% TBU gel.