Category Archives: Molecular Biology

pBR322 Digestion with EarI

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.

PCR reaction results – Succesful

I took the following gel image on my phone and used photoshop express to enhance the contrast. All gel reactions worked. Note the smudge is a reflection of light off my filter on the illuminator.

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pALS PCR for new Unzipping DNA

I’m going to try to make more unzipping DNA in one final push for my PhD. Here goes nothing, I’m all in now!!!

3-piece ligation: EpBR, BpALS, 5′-bio/int-bio adapter results

Lane assignments:

  1. 1kb ladder
  2. 5′ bio adapter reaction
  3. 5′ bio adapter reaction
  4. int-bio adapter reaction
  5. int-bio adapter reaction

I had to split each reaction into 2 lanes because the volume of the reaction wouldn’t fit in 1 lane.
image
So this reaction failed. I’m guessing the digestion of pALS didn’t work. There is an interesting artifact in this gel though. In lanes 2 and 3 you can see 2 bands between 2 and 3 kb. I have no idea why the DNA would separate like that. There are literally only 3 pieces of DNA in here: a ~30bp piece which wouldn’t be visible in this gel, a 2.5kb piece which is the EpBR fragment and is where the 2 bands are, and the 4kb pALS piece which is above these dual bands.

Oh well, I’ll try again at a future date when I run out of the DNA that I have currently produced.

3-piece ligation: BpALS, EpBR, 5′-bio/int-bio adapter

A few days ago I did a 3-piece ligation that didn’t turn out so well. Today’s ligation is more like the traditional ligation that I was supposed to do with the exception that I’m using the pALS anchor instead of the pRL anchor.

I mentioned earlier, that BpALS (pALS anchor digested with BstXI) is better overall because it is longer and makes the optical tweezer operations a little easier and better.

pBR322 can be digested with EarI or SapI for use in our construct for DNA unzipping. I haven’t done any studies that show which method produces better results in terms of final yield, but I’m pretty convinced SapI will because you only have to perform one gel extraction. If you digest pBR with EarI you get two linear fragments. And according to separate analysis by Koch and myself the overhang that we need for the unzipping construct ligation is the longer piece (in the gel image linked above it is the brighter top band).

The benefit to following this method is you can perform a three piece ligation (essentially ligating the entire construct together in one shot) because the EpBR piece (pBR322 digested with EarI) can only ligate to the adapter, and the BpALS/BpRL piece can only ligate to the other end of the adapter (both adapters that I’ve been using work the same way).

And so today I’m doing the proper three-piece ligation reaction. The reaction setup and method can be seen in the table below:

pALS PCR digestion with BstXI

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:

pALS PCR 7 (titration) results – SUCCESSFUL

image

Hooray! While the gel image isn’t the best picture ever (thanks phone), the good news is that the PCR reaction worked for every MgCl2 concentration and seems to work best at 3.5-4.5mM. Also it should be noted that lane 3 contains a visible band, but there was some smudge on the filter so it blocked the light from that lane. Here are the lane assignments:

  1. 1kb ladder
  2. 1mM MgCl2
  3. 1.5mM
  4. 2mM
  5. 2.5mM
  6. 3mM
  7. 3.5mM
  8. 4mM
  9. 4.5mM
  10. 5mM

I have purified the reactions and according to the nanodrop, I have about 11ug of DNA which translates to 87nM (233ng/ul) of tetherable DNA.

I found some EpBR (EarI digested pBR322 and gel extracted) so I’m going to digest some of this new pALS DNA with BstXI, purify, and then ligate with both adapters. If all goes according to plan, I’ll have all the unzippable DNA I’ll ever need!