Kun:LabNotes/ASE/2007-11-5

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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.