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.