Using the Fluorometer to Quantify dsDNA

The fluorometer allows accurate quantification of dsDNA. This protocol will make the quantification process run smoothly in Dr. Richard Robison's lab. Protocol has been changed based off the Quant-iT Picogreen assay protocol.

Step 1 Materials

Reagents Located in DNA Freezer
1. DNA standards
           a. One tube from the 2.0ug/mL box
           b. One tube from the 0.2ug/mL box
2. Picogreen box

WARNING: Picogreen is light sensitive; make sure to minimize exposure to light.

Items Located in Molecular Hood
1. Micro-centrifuge rack (x2)
2. 600ul sterilized eppendorf tubes
3. 100ul glass cuvettes
4. 50ml sterilized 1X TE buffer
5. 10ul; 100ul; & 1000ul reagent pipettes
6.  2;10ul; &100ul DNA pipettes
7. 10ul, 100ul, &1000ul pipette tips
8. Thin black sharpie marker
9. Small container labeled "Pico"
10. Waste container

Items Located Outside of Molecular Hood
1. Vortex
2. Centrifuge
3. Nitrile or Latex gloves (x2)
4. DNA samples to be quantified
5. Fluorometer lab notebook & pen
6. Fluorometer
7. Computer with flurometer excel sheet

Comments

[kelseymh]

This seems pretty cool! You're using Instructables as a way to document your lab procedures? Are you a grad student, a post-doc, or research staff?

The Instructables site is "intended" for anybody to be able to use the I'bles posted themselves to reproduce a project. Clearly, this one has a fair amount of detail specific to your PI's lab. However, I think it is still a great demonstration of how real molecular biology is done.

In the interests of the site's membership, would you be willing to add some details, such as hyperlinks to the fuorometer's specs, maybe to Wikipedia articles on some of the technical terms you use (such as dsDNA, which I didn't know was used to distinguish duplex from single-stranded), and so on?

For the actual project, and to help your students, you might consider having someone take photographs of you performing some of the steps. Visual cues can often be more instructive, and better understood, when they accompany written directions.

I realize this adds to your work, but it would also make this document more accessible to the much wider audience that you've exposed it to :-) Welcome to Instructables!

[robisonlab888]

I'll consider it. It's a work in progress right now.

[robisonlab888]

I know the philosophy of instructables. I wrote this for an English assignment targeted towards individuals who work in the lab with me. Thanks for the suggestions.

[isacco]

Hi woanshih , welcome to instructables community and congratulations for this tutorial. I am a geneticist and I know what explaining molecular biology to beginners means.
I agree with kelseymh. This instructables is very detailed and clearly explained, but it is far from the phylosophy of Instructables. Firstly, you did not state why you prepared this instructable. Secondly it contains a detailed list of all the steps, but it lacks some general information that could make it useful for the community.
Thanks to this detailed protocol, a layman could perfectly carry out the procedure, but without understanding its meaning and usefulness. On the other hand, a specialist would be more interested in tips and tricks that are not reported in the instrument user guide.
I suggest to add some general facts to allow non specialists to appreciate this ible. For example, why DNA need to be quantified, what is the principles of a fluorimetry assay, pros and cons, eccetera.