User:Anthony Salvagno/Notebook/Research/2010/04/19/Preplanning for Project Lambda
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I need to look through the supplies I have and get my thoughts composed for how to go about a reaction I have never done before and with no guidance outside of some web protocols and whatever the product sheets say. I also want to make sure I have everything ready like buffers, reagents, enzymes, etc. Finally I need to know how to use the QD conjugate kit. Someone misordered what I asked for and now I have to bind the avidin to one of the qd's on my own. Oh well.
Klenow Fragment, dNTPs and Lambda DNA Info
- Lambda DNA - I have 250ug of DNA in 500ug/ml solution which means I have .5ml of DNA. Nice. I'll have to figure out how much I need for a reaction. No special instructions written.
- dNTPs - 100mM concentration of each nucleotide or 25umol. No special instructions. I'll have to figure out how much of each dNTP I'll need per DNA molecule and then base the final amounts on how much DNA I'll be using.
- Biotin-14-dCTP - 0.4mM of the nucleotide in solution for an amount of 50nmol. I have to remember the differences of mol and M because right now I can't think of it.
- Klenow Fragment (-exo) - 200units at 5000U/ml is 40ul of Klenow. According to its sheet, it only requires the supplied buffer and dNTP's. One unit is the amount required to convert 10nmol dNTP to DNA in 30min at 37C.
- Neutravidin - The spec sheet doesn't say much but here is what I have:
- specific activity - 11-17ug biotin bound/mg protein
- solubility - 10mg/ml in ultrapure water
- I don't really know what to do with the Neutravidin so I'll rely on Koch when it comes to testing the reaction.
So I have qdots and an incubation buffer and no instructions. I emailed Invitrogen to try and get some information from them. I will post their reply here when I get it. Check this out.
<html><iframe height="500px" width="800px" src="http://biofizika.aok.pte.hu/en/research/nanobiotech/protocol/DNA_Biotinylation.pdf"></iframe></html>
I will use some form of this protocol once I figure out the math and all requirements.
I'll do more preplanning tomorrow, but for now here is an outline of what I'm going to do:
- Klenow Reaction
- mix reagents
- run at 37C for 30 min
- maybe heat inactivate - I don't think this is necessary but it is easy to do.
- 75C for 20 min - is it just me or is it funny that heat inactivation is almost as long as the reaction itself
- EtOH precipitate
- Bind Neutravidin to Klenow Product
- Run Gel of Bound product vs unbound (Neutravidin vs No protein)
- Either do reaction again or use unbound product and attach conjugated QDs.
- Give to collaborators for experiment
- Steve Koch 23:02, 19 April 2010 (EDT): I would also plan on seeing whether you can make QD "couplets" with your DNA. It may be a striking and easy way of determining success. You'll also need to find out the optimum way of running lambda DNA on a gel. What percentage, etc.