Yay, good digestion! Here is some useful information about which piece to gel extract. I’m always looking for this mostly for doubling checking purposes, but I need to figure out a place to store this so I can EASILY find it. For now all that matters is I cut out the larger band (top) and do gel cleanup. Onward and upward I suppose…
I extracted into 2 tubes and here are the final amounts:
- 88.7ng/ul –> 52nM
- 101.9ng/ul –> 60nM
The digestion is complete. There is really no way for me to verify the results so I just went ahead and did an enzyme cleanup and purified the DNA. Now I have:
- Tube 1 – 170.0ng/ul –> 63.75 nM
- Tube 2 – 178.3ng/ul –> 66.86nM
Once I verify the pBR322 digestion and gel extract (coming up next) I can set up the ligation reaction and finish up!
Since all the reactions yesterday worked, I purified the DNA and took some measurements in the nanodrop:
- Tube 1: 289.6ng/ul in 30ul –> 8688ng
- Tube 2: 259.8ng/ul in 30ul –> 7794ng
Since those yields look phenomenal, I decided to run my digestion of the anchor with BstXI. Once I gel extract my pBR322 digestion, I can run the final step, LIGATION! I’ve never completed this process in 2 days, and I hope I didn’t just jinx myself. Anyway, here is the digestion of pALS:
This is the DNA that we unzip. First we need to cut it and gel extract it since EarI cuts in two places on the plasmid.
The pALS anchor is special in that it has two purposes. The first is that it is immediately usable in DNA stretching experiments because it has a dig molecule (to stick to glass) on one end and a biotin (to stick to microspheres) on the other. The second is that I can digest it with BstXI restriction enzyme and use it as the anchor segment for unzipping DNA.
And when it all works well, pALS is much more useful than the pRL574 anchor (1.1kb). It’s extra length makes it easier to calibrate the optical tweezers for unzipping and we can get higher forces in the optical trap by using bigger beads. (Note: The tweezers are the entire device, and the trap is the focal point of the laser in the microscope. So the trap is a subset of the tweezers.)
With the huge success of the pALS PCR yesterday, I’m going to digest some of it and then ligate this piece to EpBR and both adapters. But first here is my digestion reaction: