Ligation Try 2

Thursday’s ligation failed. So today I’m trying to adjust. Based on Bill Hooker’s suggestion, reducing the amount of ligase could be a solution so I’m trying that. I’m also increasing the ligation time between each adapter addition to give the reduced ligase time to ligate. Finally I’m adding a third reaction using the leftover DNA from Thursday (concentration of EpBR is 60nM and BpALS is 67nM). Check out the reaction below.

  • bill

    A few more ideas —
    What’s the rationale for the stepwise addition of adapter?
    Would it be worth setting up vector-only and adapter-only reactions to check for unwanted concatamerization?
    What’s “old” in this context? — DNA that has been stored a long time and/or frozen/thawed a lot will degrade, esp. sticky ends, which can fuck up your ligation
    Transformation into competent cells is more sensitive than staining a gel — that is, you can have a successful transformation from far, far less DNA than it takes to give a visible band on a gel.

    • Anthony Salvagno

      Transformation is not an option in this case. The anchor piece I refer to is dig-labeled, and the adapter piece is biotinylated. The adapter is also old. It hasn’t been through a lot of freeze-thaw cycles. Maybe like 5. But I have one adapter duplex (~25bp) that is from 2009 (that’s the “really old” one, I’ll need to find it in my OWW notebook), and one from 2011 (that’s the “old” one). There is also a nick between the adapter and the anchor sequences, which needs to be carried through. Finally the ligation is run through a gel and then the ligation product is gel extracted for DNA tethering experiments and DNA unzipping (hopefully).

      In the past I have done anchor only ligation, and unzipping DNA only ligations. I’ve never gotten results that are any different than this method. The 3-piece is just a bit of a time saver. And generally once I get the 3-piece ligation to work then I know the reaction works and I will experiment with the other technique, because I feel it is more efficient and less prone to contamination (I hate gel extraction because of potential agarose residue).

      The rationale for the stepwise addition of adapter is so that I don’t end up with adapter ligated to both the anchor and the unzipping piece. Both those pieces have non-palindromic sticky ends with different sequences (anchor is BstXI digested, and unzipping is EarI digested). So if I put in too much adapter I’ll end up with non-complimentary sticky ends. The idea is to start low and let the adapter ligate where it will and then give it time to make the connection to the third piece. I hope I’m explaining this well enough.

    • Anthony Salvagno

      Man I wish I could clone this construct. I would have been done with this project years ago!