Category Archives: Shotgun DNA Mapping

pALS BstXI digestion results

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!

PCR yield and BstXI digestion

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:

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.


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!!!

Dissertation Plan

Here is my dissertation and defense plan. My research is composed of three areas:

  1. Shotgun DNA Mapping
  2. D2O Effects
  3. Open notebook science

All of it will be tied together because of ONS and D2O. Let’s get to it!!!

Create your own mind maps at MindMeister

DNA Overstretching Data

For the past few days, Pranav’s been hard at work collecting DNA tweezing data. The first set of experiments revolve around DNA overstretching (which I’ll explain later, but you should feel free to google it). We’re planning on analyzing force differences between DNA in D2O and H2O through overstretching. Hopefully eventually we’ll be working towards DNA unzipping, but that is less likely given our time constraints (graduation!!). Regardless there are a lot of interesting studies to be done here.

DNA overstretching, several force profiles are overlapping. Notice the repeatability.

Here is the link to the notes. And here is the folder where all the data is stored.