It looks like the ligation didn’t work exactly as planned, but I would say definitely better than the 3 piece ligation debacle. I also let the gel run for too long, so the 1, 1.5, and 2 kb bands have run off the gel. The bottom band in the gel is the 3kb band. The brightest bands in my gel are the 4.3kb linear pBR322 fragment and then what would be the ligation product. And it appears the ligation didn’t work at all in the 5’bio reaction from this morning. Oh well gel extraction and unzipping attempts are next on the “To Do.”
After my earlier reaction it is time to continue on. I have purified the ligation reactions from before to remove enzyme and unligated adapter molecules (QiaQuick cleanup kits work really well for purifying short DNA molecules). I ran some samples in the nanodrop to calculate the resulting concentrations (shown in the reaction below, and calculated from the DNA concentration calculator). Next I’m running the reaction below:
5pBR – pBR cut with SapI and ligated to the 5′-bio adapter
IpBR – pBR cut with SapI and ligated to the internal-bio adapter
pRL anchor – 1.1kb PCR fragment obtained from pRL574 and cut with BstXI
So yesterday’s experiment had some interesting results. Today I’m trying a different ligation approach. Instead of doing a 3-piece ligation, I’m doing two separate ligations: the unzipping DNA to the adapter, and then this product to the anchor. Here is the nomenclature for the reaction shown below:
SpBR – pBR322 fragment digested with SapI restriction enzyme
5′-adapter – adapter DNA molecule with biotin attached to the 5′ end
internal adapter – adapter DNA molecule with biotin attached to a dT within the molecule
In keeping with my naming scheme, the products of these reactions will be labeled 5pBR and IpBR, for 5’adapter-pBR and internal-adapter-pBR respectively.
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!
I skipped a whole bunch of steps in the process, but I discovered some prepared DNA from last year that I could use in what is normally the final reaction to make unzippable DNA. The normal process is:
create the anchor via PCR
digest the PCR anchor with BstXI
digest pBR322 with SapI/EarI (EarI creates two fragments which requires a gel extraction, while SapI creates one long fragment)
3-piece ligation with anchor, unzipping fragment, adapter
The DNA I found I named BpALS, BpRL, and SpBR. The nomenclature is a little strange but the B stands for BstXI, which means those DNA pieces (BpALS and BpRL) are digested with BstXI. The S stands for SapI which means SpBR has been digested with SapI.
Today I set up a ligation reaction using this old DNA (since DNA is really stable and stays usable for long periods of time) and using the new adapters I purchased. Check out the reaction below:
This reaction is different than a normal ligation reaction. In this reaction I start with a small concentration of adapter compared to the concentrations of anchor and unzipping DNA and slowly add more adapter. The reason for this is because I want to ensure that the ligation creates one continuous piece. If I add equal concentrations of all three pieces, then I could end up with a lot of adapter ligated to both the anchor and the unzipping DNA. Since the adapter is designed to not ligate to itself this would create two fragments that cannot ligate to each other.
NOTE: There is a really good chance I sabotaged this experiment. Digesting pBR322 with SapI produces a singular and linear DNA product because it cuts in exactly one location. The problem is that when ligating, this may self ligate. I normally do a ligation with SpBR and the adapter with the adapter in tremendous excess to prevent this. Then I purify that reaction and do another ligation with this new product (that I call ApBR for adapter-pBR322) and the anchor piece. Today I forgot to do this first ligation reaction and went straight for the three piece method which was designed for the EarI-digested unzipping fragment otherwise known to me as EpBR. Hopefully the gel analysis reveals some good news.