Category Archives: Molecular Biology

Ligation, Molecular Biology, Shotgun DNA Mapping

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

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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.
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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.

Ligation, Molecular Biology, Shotgun DNA Mapping

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

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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:

Digestion, Molecular Biology, Shotgun DNA Mapping

pALS PCR digestion with BstXI

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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:

Molecular Biology, PCR, Shotgun DNA Mapping

pALS PCR 7 (titration) results – SUCCESSFUL

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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!

Ligation, Molecular Biology, Shotgun DNA Mapping

Tetherable DNA concentrations

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I finished the gel extraction from the ligations of yesterday. So I decided to spare the 1ul required to measure the concentrations of the DNA for the nanodrop. I also measure the samples from the 3-piece ligations. Here are the resulting concentrations:

  • Some nomenclature:
    • T#pXX: T stands for tetherable; #indicates which adapter is in the final product (either 5’biotin or internal-biotin); pXX is the plasmid name for the adapter (either pRL or pALS)
  • T5pRL (~5.4kb)
    • 23.8ng/ul
    • 6.6nM
  • TIpRL (~5.4kb)
    • 9.3ng/ul
    • 2.6nM
  • T5pRL (~5.4kb, 3-piece result)
    • 14.7ng/ul
    • 4.1nM
  • TIpRL (~5.4kb, 3piece result)
    • 50.4ng/ul
    • 14nM
  • T5pALS (~8.4kb)
    • 23.6ng/ul
    • 4.3nM

Overall a much better yield than I expected, but I don’t completely trust the nanodrop so take these results to be whatever you want. In my experience I’ve never had good tethering efficiency with anything less than about 100pM DNA so I would recommend dilution of each of these about 1:10 for tethering purposes.

Ligation, Molecular Biology, Shotgun DNA Mapping

SDM 2-piece ligation results

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Here are the lane designations:

  1. 1kb ladder
  2. 5’bio reaction from yesterday
  3. 5’bio reaction from this morning
  4. internal-bio reaction from yesterday
  5. internal-bio reaction from this morning

image

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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.”

Molecular Biology, PCR, Shotgun DNA Mapping

pALS PCR 7: Mg++ Titration

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Time to optimize the PCR reaction so that I can be sure the reaction will work every time efficiently. This way I can just do a few more PCR reactions and give myself a good supply of 4kb anchor to work with from here on out. Here is the reaction:

Ligation, Molecular Biology, Shotgun DNA Mapping

Ligation: 5pBR and IpBR to pRL anchor

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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

Ligation, Molecular Biology, Shotgun DNA Mapping

Ligation: SpBR to 5′-adapter and internal-adapter

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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.