So the good news is that had I not thoroughly messed up the reaction, this would have been a resounding success. The other good news is that this part of the experiment is fairly simple to repeat using the pRL574 anchor. The bad news is the ligation went as I expected it to which is to say sloppily.
Now I have to try to explain to you these results:
Above is an image of the gel under UV light stained with Ethidium Bromide. I used to use Sybr Safe for visualization (and have in the first part of these experiments), but in the past unzipping was never successful so we think there is a chance that Sybr Safe interacts with DNA differently than EtBr and could hinder unzipping. So in an effort to identically repeat Koch’s grad school work I’m using EtBr because that is what he would have done.
Below is a cropped and grayscale version of the above image:
We are looking at a lot of lines here. There is a meaning to them all though:
- The two outer lanes are 1kb ladders in different amounts. More specifically the right most lane has twice as much ladder as the left most lane. We’ll call these lanes 1 (left) and 5 (right).
- The three middle lanes from left are: ligation with 5′-bio adapter (lane 2) and pRL anchor, internal-bio adapter and pRL anchor (lane 3), and 5′-bio adapter with pALS anchor (lane 4).
- The ladder reads (from the bottom): 1kb, 1.5kb, 2kb, 3 (brightest band in the ladder), 4, 5, 6, 8, 10kb
- Lanes 2 and 3 are identical length wise and the only difference between the molecules is where the biotin molecule is located.
- The band at the very bottom is the pRL574 PCR product band which is 1.1kb in length.
- The next two bands above that are pBR322 that has religated into circular DNA.
- The band above that is digested pBR322 (4.3kb).
- And the band above that is the unzipping DNA that we want, which is around 5.4kb. I have gel extracted that piece.
- All bands above this band are chains of pBR322 that ligated together and may or may not have the anchor and adapter ligated to it.
- Lane 4 is interesting. I would be inclined to say that we got better ligation out of this band because there is less circular DNA bands (compare with lanes 2 and 3). But the product is harder to quantify.
- The brightest band is the pALS anchor, 4kb in length.
- Barely above that is the digested pBR DNA, 4.3kb in length. And is identically positioned with the other two lanes.
- Above that is a very feint band and now I’m inclined to believe that this band represents two pBR322 fragments ligated together and circularized. (Above I say its the product of the reaction that I wanted). I think this because this band lines up exactly with bands in the other two lanes.
- The next visible band I believe is the product that I expect, 4kb pALS + 4.3kb pBR -> 8.3kb unzipping sequence. It is right there, but again bands in the other lanes could indicate something else.
Well tomorrow I’ll gel extract and hopefully we can get some DNA tethers by the end of the week and try for unzipping. Also tomorrow I’ll retry the ligation reaction for pRL-pBR (since I have no more predigested pALS). Tethering is the only real way to identify successful ligation so we’ll find out together!