Biomod/2011/Caltech/DeoxyriboNucleicAwesome/Protocols

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Protocols

Things that need to be written

  • Quantitating DNA
  • How to run a gel
  • How to do AFM (when we learn this)
  • How to analyze fluorescence experiments (when we learn this)
  • How to perform gel purification
  • How we ran experiments...


Gel Purification

  • 1. Anneal the complex (Q-function)

For 300ul sample, add 50ul BB_ND dye 170ul of sample will be loaded per lane. Samples will take 2 lanes, and one blank lane should be placed between different samples.

  • 2. Wash the big plates. Dry the plates for 45 minutes

- Feel it with fingers/ Watch out for the water flow

  • 3. Make a gel. 12% ND gel for purification

Lulu’s recipe for 40ml of 12% ND gel which works for one gel - 12ml 40% Acrylamide - 4ml 10x TAE/Mg++ - Fill up to 40ul MQ water (add 24 ml) - 240ul 10% APS, slow mix - 24ul TEMED, slow mix 

  APS should be no more than 10 days old
  • 4. Run the gel for 150V, roughly 4.5 hours ~ 6 hours (when the dye ran off the gel) depending on the size of the sample. For example, if the sample is 122nt~176nt, run it for 6 hours. Change the buffer in every 1.5 hours.


SPEX protocol

  • 1. Turn on the lamp (back side). Wait for 30 min

Bottom big on/off button is for the machine. It is always on. Top small one is for the lamp. If there’s somebody who will use the SPEX tomorrow, then do not turn off the lamp after the experiment.

  • 2. Turn on the computer if it’s not on.
  • 3. Software: Instrument control center

Default: Click OK

  • 4. Temperature set up

Click the third square icon called “Visual setup” Click the middle square Temperature control icon: Turn on and Set to 25 degreeC Choose T bath (internal). NOT Probe (external).

  • 5. Wash the cuvettes.

Be careful! Hold only top or bottom. DW (10 times) 70% ethanol (twice) DW (5 times) 70% ethanol (once) While you wash, you can touch the side. Hold it in a stable way. Let it dry (upside down)

  • 6. Dry the cuvettes using lens paper.
  • 7. Click second square icon called “real time display”.

High voltage: HV on, S 950, T 950 Bandpass : Set 2 for everything

  • 8. Click the last square icon called “run CWA”.

Open a file called “ROX_CWA” from e:\SPEX DATA\biomod Optional mode: kinetics, Integration time: 10s Define: Repeat acquisition: 1 min (minimum) for total of 180 min (3 hours)

  • 9. Mixing: Hold top. 350ul of 1000ul pipette all the way in. Mix it for 30 times.
  • 10. Put the cuvettes. Logo faces out!
  • 11. Start acquisition. Run sample.

Range of the real data should not exceed 1 million (1000,000) Number we see is S/R  will be (100 million)

  • 12. Add more samples in the middle of SPEX experiment

When the status is “waiting”, click “stop”. Don’t click anything else. Add and mix. After you put the cuvettes inside the box, click “no”.

  • 13. Save the data

When the status is “waiting”, click “stop”. Click “yes” Save data in two forms (DWA datafile, multi~ test) Check if the data is properly saved: Check the last data.

  • If you want to see the reference, go to options: view option and display raw R.

How to Open SPEX Data in MATLAB

  • Open up MATLAB and use File->Import Data
  • Navigate to the raw-text (.txt) data file and open it
  • The table on the right should show eight rows, where the odd ones are the times associated with the signal from the row under them. The top set is sample 1; the next is 2, the next is 3, the bottom set is sample 4. Press "next."
  • Optionally right click on "data" and rename it to whatever you want. This isn't necessary unless you're afraid of your data getting overwritten eventually when you import another set.
  • Click "finish."
  • Your data should be visible in the "workspace" window with the name "data" or whatever you might have renamed it to. Right click on it -> "open selection" to view it.
  • To plot a single sample's data, use this, where "data" is the name of your data variable:
<syntaxhighlight lang="matlab">plot(data(1, :), data(2, :))</syntaxhighlight>
  • To plot all four samples' data on the same plot, use this:
<syntaxhighlight lang="matlab">plot(data(1, :), data(2, :), data(3, :), data(4, :), data(5, :), data(6, :), data(7, :), data(8, :))</syntaxhighlight>
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