I've got two gels running while I type. Both will help me decide if the MAP7 DNA preps we have are suitable for my tweezers experiments. Both gels contain high and low concentrations of two different MAP7 DNA preps, along with a size standard consisting of intact phage lambda DNA (48.5kb) and a HindIII digest of the same DNA.
The first gel is a conventional agarose gel - the voltage is created by a pair of simple wire electrodes, one running across each end of the gel box. To increase the resolution (i.e. separation) of DNA fragments bigger than 15-20kb, the gel has a lower concentration of agarose than is usually used (0.6% rather then 0.8-1.0%). This makes it more fragile, so I'll need to handle it very carefully tomorrow when I'm photographing it. I'm also using a much lower voltage, which will also help spread out the big fragments and squeeze together the little ones I don't need to resolve.
The second gel is a "CHEF" pulsed field gel. This uses pulsing electric fields in different directions (120 degrees to each other) to jiggle the DNA fragments back and forth as they move through the gel. This forward-left then forward-right pushing has little effect on the separation of small fragments (less than about 20kb) but it greatly improves the separation of the big fragments. The new apparatus (belonging to the new lab next door) is much more sophisticated than the old one that's collecting dust on our top shelf, and I had to call technical support to learn how to dumb it down enough that I could control what it was doing.
In admiring this new apparatus we developed a new rule of thumb relating the cost of a piece of scientific equipment to the number of buttons it has. This one has more than 50 buttons, and cost about $40,000; our old one had about 7 buttons and cost $4500. This only applies to equipment that doesn't come with its own computer. The real-time PCR machine we share with other labs cost $70,000; it gets away with having only a single button because it's controlled by very complicated software.
Field of Science
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Change of address1 year ago in Variety of Life
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Change of address1 year ago in Catalogue of Organisms
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Earth Day: Pogo and our responsibility1 year ago in Doc Madhattan
Showing posts with label DNA. Show all posts
Showing posts with label DNA. Show all posts
Not beads-on-a-string but strings of DNA on a bead
The other project I'm taking on is the controls and preparation for our laser tweezers analysis of DNA uptake. Our previous work (really the physics grad student's work) was trying to get cells to attach to and take up a specific bead-attached DNA fragment with a single USS. If this had worked we hoped it would let us study how DNA uptake depends on the orientation of the USS. We now realize that this was too ambitious an initial goal.
Our new experimental goals are to study the forces that act on the DNA during uptake, in wildtype cells and in cells with mutations in specific uptake proteins. This doesn't require use of a defined DNA molecule with only one USS, so we can change the setup to optimize our chances of success.
We also belatedly realized that we can use Bacillus subtilis as a positive control. Laser tweezers have already been used to study forces generated during B. subtilis DNA uptake. (Because the B. subtilis uptake mechanism is quite different than that of H. influenzae this serves as proof-of-concept for our experiments but doesn't make them redundant.) B. subtilis cells are larger and more robust than H. influenzae cells, and their DNA uptake does not depend on a particular sequence. Because the conditions for B. subtilis tweezer assays have already been worked out, we will make sure we can demonstrate uptake by it before moving on to uptake by H. influenzae.
The new plan is to first make sure we have lots of DNA fragments attached to each polystyrene bead, before trying to detect uptake by either B. subtilis or H. influenzae. My first step is to get a preparation of randomly broken H. influenzae DNA that consists mainly of fragments 50-100kb long, and then to attach these fragments to the beads by sticking biotin on the ends of the DNA, and using beads pre-coated with streptavidin, which binds tightly to biotin.
I've decided on 50-100kb for several reasons. First, it's hard to work with fragments longer than this, because longer fragments break unless they're handled very gently. Second, it's easy to get fragments about this size simply by not being gentle with the DNA prep. Third, this will let me attach a LOT of DNA to each bead. Fourth, the individual fragments will be substantially longer than the cells and the beads: the beads are 1 micrometer in diameter, the cells are 1-3 micrometers long, and a 60kb DNA fragment is about 20 micrometers. But they aren't so long that they extend a long way away from the bead they're attached to.
I'll use DNA from strain MAP7, partly because we already have lots of it (made by one of the post-docs) and partly because it carries antibiotic-resistance alleles that we can use to detect whether cells have taken it up. This will be important in checking whether the beads really do have DNA attached to them.
The DNA in the prep we have may already be broken into appropriately-sized fragments. I can check for small pieces by running this DNA in a normal agarose gel; fragments bigger than about 25kb will all jam up at the top of the gel, and only smaller fragments will spread out in the gel. A better check for DNA size will be to run the DNA in a pulsed-field agarose gel, whose rapidly reversing electrical current allows even very big fragments to spread out in the gel. Luckily our neighbours in the lab have a new apparatus to run these gels. (We have a 15-year old system which I suspect no longer works.)
If this analysis says the DNA fragments are too small, I'll make a fresh prep, handling it more gently. If the analysis shows that the DNA fragments are too big, I'll just rough up the DNA prep by whirling it in the vortex mixer, or by forcing it through a narrow syringe needle.
The next step will be adding the biotin to the ends....
(Hmm, the spell-checker thinks that tweezers must be plural; it doesn't approve of "laser tweezer analysis".)
Our new experimental goals are to study the forces that act on the DNA during uptake, in wildtype cells and in cells with mutations in specific uptake proteins. This doesn't require use of a defined DNA molecule with only one USS, so we can change the setup to optimize our chances of success.
We also belatedly realized that we can use Bacillus subtilis as a positive control. Laser tweezers have already been used to study forces generated during B. subtilis DNA uptake. (Because the B. subtilis uptake mechanism is quite different than that of H. influenzae this serves as proof-of-concept for our experiments but doesn't make them redundant.) B. subtilis cells are larger and more robust than H. influenzae cells, and their DNA uptake does not depend on a particular sequence. Because the conditions for B. subtilis tweezer assays have already been worked out, we will make sure we can demonstrate uptake by it before moving on to uptake by H. influenzae.
The new plan is to first make sure we have lots of DNA fragments attached to each polystyrene bead, before trying to detect uptake by either B. subtilis or H. influenzae. My first step is to get a preparation of randomly broken H. influenzae DNA that consists mainly of fragments 50-100kb long, and then to attach these fragments to the beads by sticking biotin on the ends of the DNA, and using beads pre-coated with streptavidin, which binds tightly to biotin.
I've decided on 50-100kb for several reasons. First, it's hard to work with fragments longer than this, because longer fragments break unless they're handled very gently. Second, it's easy to get fragments about this size simply by not being gentle with the DNA prep. Third, this will let me attach a LOT of DNA to each bead. Fourth, the individual fragments will be substantially longer than the cells and the beads: the beads are 1 micrometer in diameter, the cells are 1-3 micrometers long, and a 60kb DNA fragment is about 20 micrometers. But they aren't so long that they extend a long way away from the bead they're attached to.
I'll use DNA from strain MAP7, partly because we already have lots of it (made by one of the post-docs) and partly because it carries antibiotic-resistance alleles that we can use to detect whether cells have taken it up. This will be important in checking whether the beads really do have DNA attached to them.
The DNA in the prep we have may already be broken into appropriately-sized fragments. I can check for small pieces by running this DNA in a normal agarose gel; fragments bigger than about 25kb will all jam up at the top of the gel, and only smaller fragments will spread out in the gel. A better check for DNA size will be to run the DNA in a pulsed-field agarose gel, whose rapidly reversing electrical current allows even very big fragments to spread out in the gel. Luckily our neighbours in the lab have a new apparatus to run these gels. (We have a 15-year old system which I suspect no longer works.)
If this analysis says the DNA fragments are too small, I'll make a fresh prep, handling it more gently. If the analysis shows that the DNA fragments are too big, I'll just rough up the DNA prep by whirling it in the vortex mixer, or by forcing it through a narrow syringe needle.
The next step will be adding the biotin to the ends....
(Hmm, the spell-checker thinks that tweezers must be plural; it doesn't approve of "laser tweezer analysis".)
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