Last month when I was working on the DNA project, I reported an issue with the freezer. Well that same issue has struck again only this time it has gone unchecked for two weeks or longer! Check this out:
Yes that is one solid block of ice… yea for me!
I have some anti-dig in our -80C Freezer and I’ve discovered that it is almost 3 years old. This probably isn’t that big a deal, but since I’m starting from scratch I would have let it slide if it was a year old. It’s not. So this will be ordered today.
The real reason I wrote this notebook entry was partly to declare that I’m ordering new anti-dig, but also because I was super stoked that I googled “anti-dig openwetware” and the notebook entry I linked above was the #1 hit. It’s good to know that my old notebook and new one are both making an impact in the scientific world.
This is what I’m ordering: Anti-dig
There are times when I rely on the nanodrop (that I use for Yeast/E. coli measurements) to measure DNA amounts. The machine tells me concentrations in terms of ng/ul but that number is useless most of the time so Steve created this concentration converter that will turn ng/ul into molarity (M) depending on the length of the DNA sequence being analyzed. I’m now sharing this calculator with you all. Public editing is turned on, and you should be able to create your own copy in Google Docs. Otherwise, just edit the DNA length (column labeled “length of ds”) and the concentration (column labeled “mass conc.”) and the third column will show you the concentration in nM.
DNA Concentration Calculator via Steve Koch
Download for Excel
I had to do a bit of reorganizing. Since I’ve been reordering new supplies, I can easily get confused between what I need and what I don’t. So I’m conglomerating all the things that I do into one box and everything else will get shoveled away to be lost for all eternity. Here is a picture of the items that I need immediately and what they are:
Update:
I am placing an order for the oligos and primers needed for Shotgun DNA Mapping. Here are the sequences I’m going to order:
I’m ordering from Integrated DNA Technologies. I was going to order three different versions of the bottom adapter, but decided to just get the two listed above. The one I omitted from the order is the 5′-bio with the floppy overhang (which won’t ligate to anything). Once I get my second supply of money from IMSD I’ll order that oligo.
Today I need to buy microspheres. In the first experiments we used spheres with a diameter of 0.5um (or 500nm), and over time we eventually switched to using 1.0um beads. The reason is because: 1) big beads are easier to see if they are tethered or not, 2) you get better tweezer forces with the larger beads, and 3) the big beads clump less. The only drawback to using the larger beads is that there is a lot of repulsion between the beads and our glass surface so our DNA tethers need to be longer. I invented the 4kb pALS anchor to solve this very problem.
Here are some pictures of the different sized beads in the tethering environment:
And for completeness here are some old notebook entries regarding those pictures:
And here is a video that shows the tethering results of the DNA experiments listed above:
We want beads that are coated in streptavidin (or some form of avidin) because this molecule creates a very strong bond with biotin which is attached to our DNA for stretching/unzipping experiments.Most commonly, you can order beads with streptavidin, but some companies offer alternatives like avidin or neutravidin. In my experience neither works any better or worse than streptavidin. And in the case of these experiments the bond either holds or it doesn’t.
But believe it or not, it is hard to quantify the effectiveness of the beads. As you can see in the pictures above, both bead concentration and DNA concentration can affect tethering efficiency. And I have suspicions that the sonication process (what we do to prevent the beads from clumping) may affect the streptavidin in some way: in my head the vibration shakes off the molecules from the beads.
With all that said, there are places that I trust buying beads from. In the past I’ve purchased beads from Bangs Labs, Invitrogen, and Poly Sciences. I’ve never noticed that any bead from any company seems to work better than the rest. Because of this I think I’ll order a new stock of beads from Bangs because they pretty much only make beads (so they should do it the best). Note: I just remembered that Bangs, and Poly Sciences may be the same company and it turns out they are affiliated in some way. So I suppose there really is no difference between the two.
Update: I’m placing my order with Bangs Labs. I’m ordering 0.53um beads and 1.04um beads, both coated in streptavidin, and neither are fluorescent.
I’m basically making a “site” map so I can find the forms that I’ve been emailed and filled in, in case there are issues in the future, I can find them and pull them up again. Easily.
Here are the useful items:
There is almost no reason to write this post other than to document the fact that I am finalizing my paperwork for my PhD candidacy. Please don’t clap/cheer or congratulate me. There is quite a story about why I even need this post, but just know this is mostly for my own documentation. But if you are interested, here is the full story:
From Fall 2007-Spring 2010 I was an NSMS IGERT fellow. The NSMS IGERT created a degree program from the curriculum of the program. NSMS fellows could be either NSMS PhD students or students from outside the field. If they were from another department, those students would be eligible for a minor in NSMS. I fell into this category.
Unfortunately I didn’t complete the curriculum. There were 5 courses and due to time constraints I had to drop the last course. Doing so didn’t affect my Physics degree at all, but it did affect my eligibility for the NSMS minor.
Fast forward a short time later and I gave my candidacy presentation Fall 2010. With that came a bunch of annoying paperwork. But I got it done and presented and passed and the rest is history. Right?
Wrong!
I met with the NSMS program administrator last week to discuss my IGERT proposal. In a completely chance discussion, she mentioned to me that there was a recent rule change so that I would be eligible to receive the minor in NSMS. All I would need to do is attach a form to my candidacy paperwork and I’m in.
So I contacted the Physics admin to discuss the logistics and discovered that my candidacy paperwork never got filed. I’m not sure if it never was turned into the UNM Office of Graduate Studies (OGS), or if got lost, or what. I am sure that I filled it out and submitted it.
How you ask? Well last summer I submitted paperwork to receive an MS in Physics. The paperwork for that is identical to the application for candidacy, just with a different title. And I copy and pasted all the information from the candidacy form onto the master petition.
Regardless of what went down at my candidacy exam, some how there is no record of my application for candidacy being submitted and that is what I’m finalizing today. And since I document anything from this process in my open notebook, I can’t prove that the error was not my own. But now I am documenting it, so if there are any issues in the future I can point here and figure out what the issue is.
See, open notebook science works well for other things than just science.
And in case anyone is interested, here is a practice candidacy exam I created complete with audio:
I did a mini-study this morning to find out what the minimum volume needed is to get an accurate reading in the nanodrop. I have 2 different cuvettes (semi-micro and micro) and I wanted to impact the cultures the least so I would like to use the micro cuvettes.
I used 5 semi-micro cuvettes and 2 micro cuvettes. I put increments of 100ul starting at 100ul in each cuvette (100 -> 500ul in the semi-micro and 100 and 200ul in the micro cuvette). At and above 400ul the nanodrop was able to effectively and consistently read the absorbance of the semi-micro cuvette (verified because the readings for 400 and 500ul were identical). For the micro cuvette, I looked at the profile (image above) and saw by eye that the height of the meniscus of the liquid media was roughly equivalent to the height in the semi-micro cuvette with 500ul. So I put this in the nanodrop and got an equivalent absorbance reading.
So in summary:
The End.
I’ve been slacking with keeping up with the equipment that I use in the lab, but I have updated the product page and it should be all up to date with the latest string of experiments. Let me know if you notice I’m missing something.