User:Anthony Salvagno/Notebook/Research/2010/05/10/Project Lambda Phase 1 Part 2
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Last time I ran the Klenow reaction and started purifying. I left off at the ethanol precipitation stage. So I just need to spin the pellet down and remove the ethanol. Next I need resuspend in buffer I will use just T buffer. After that comes the next phase of experiment.
Plan for today
I have to go to main campus to sign some IGERT stuff and then I will get to work. But here is the plan for what I need to do:
- Resuspend with 20ul of T buffer. Then put about 5ul of DNA solution into new tube.
- Add Neutravidin to the 5ul tube. I will need to figure out how much I should use.
- Prepare a gel.
- Put pure DNA and neutravidin bound DNA in columns and run the gel.
- Determine next step after analysis:
- I will either need to start over if the gel is a failure
- Talk with Koch if the results are inconclusive
- Plan for qdot binding and visualization.
I ran a nanodrop reading of the purified DNA. I got 320ng/ul which isn't bad from the starting amount of 500ng/ul. This gives me 9.8nM DNA. I'll just have to do some reading with the neutravidin. I labeled the tube lamda-FI (for fill-in).
- diluted to 10mg/ml with water, needs to get to 1mg/ml in PBS
- 5mg/ml is 83uM
- From my own calculation on google I get ~33uM ((10*10^-3/(60*10^3))/(5*10^-3)
- mol weight 60kDa
I am going to run 4 lanes:
- lambda DNA no protein no fill-in
- lambda with fill-in
- lambda with neutravidin
- I want around 40nM of this stuff and 1mg/ml is 16uM so I need to dilute my neutravidin 1:400. Of course first I need to dilute what I have (10mg/ml in water) to 1mg/ml in PBS and then dilute again but with T buffer since that is what I have the DNA in.
- lambda with QDs bound
- I want a final QD concentration greater than 20nM since I have 9nM DNA but there are 2 ends per DNA molecule so double the concentration of available binding sites. According to the specs and my notes I can dilute up to 40nM so I will do just that.
- I want 40nM of 525-QD (since the concentration is 2uM) so that is 49ul of the incubation buffer for a total of 50ul.
The gel melted or something so I'm going to redo this experiment tomorrow.