User:Anthony Salvagno/Notebook/Research/2010/02/04/Trial 7.0 Results
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Results
Ok I think I can safely and officially end this charade! Ok, it's not a charade, but I think using Osley's PCR has taught me something. The protocol works, but that amount of Cy3 in solution royally messing up a gel reading. The only thing available to me now is to PCR cleanup and run another gel to see what happens.
If you look at the image, Cy5 definitely worked at all ranges of MgCl2. There is a smear, but that is what happens when you run protein on a gel from my experience. I would venture to say Cy3 PCR also worked at most (maybe not all) concentrations of MgCl2, but it is so damn fluorescent that it is impossible to tell. Like I said a paragraph ago, I will PCR cleanup and then run another gel. I will also nanodrop all the samples so that I can get an accurate measure of how much DNA is in there and how much fluorophores are in there. I think I can say job well done and give myself a good ole pat on the back though!
SJK 18:25, 4 February 2010 (EST)
Woohoo! Congrats! I agree that there's for sure successful PCR on the right side, and probably on the left side too. The new format for showing the gel is fantasic, BTW. Also, I think we need to do that same thing to older gels and make a real assessment of whether Osley machine is better or not. Glad you got it working!
- Ramalldf 23:13, 4 February 2010 (EST):That's cool, congrats man!
- Steve Koch 23:59, 4 February 2010 (EST): Also, I now realize this was an unforseen peril of SYBR safe. W/ Ethidium, would be using UV illumination and those labels probably not a big deal.
Andy's comments
Andy Maloney 01:22, 5 February 2010 (EST): So I have been curious about the interaction of Sybr Safe, Cy3 and Cy5 since we got the first gel images. I downloaded the spectra for Sybr Safe, Cy3 (attached to an IgG), Cy5 (attached to an IgG) and the Safe Imager manual from Invitrogen and plotted their spectra. I should note that it looks like the Cy3 and Cy5 dyes don't change their spectral characteristics when attached to the IgG.
It looks like the transilluminator does excite some Cy3 which in turn will excite the Cy5. One interesting thing to note about the Sybr Safe is that it can be excited in the UV so no need for the ethidium, just an UV transilluminator.
- Steve Koch 01:56, 5 February 2010 (EST): Interesting. I didn't know that about the SYBR safe / UV, or maybe I'd forgotten. I guess it makes sense, otherwise they'd never have broken into the market. I think we probably have money to buy a nice UV transilluminator. Ant, you should probably look into that. Needs to be <$2,000, which I think is easy to do.
- Andy Maloney 08:50, 5 February 2010 (EST): Oh wait. No. It was late and my brain wasn't working. No matter what if we use the Sybr Safe stuff, it will always excite the Cy3 because the emission of the Sybr Safe overlaps with the excitation of the Cy3. Also, I'm pretty sure that the peak in the UV for the Sybr Safe dye is an absorption peak and not an excitation peak.
- Steve Koch 09:18, 5 February 2010 (EST): Actually I still think you were right. Even though SYBR emission overlaps with Cy3, that excitation will be far far less efficient than the bright-ass illumination with the blue light illuminator. So, I think it would look much better under UV. Furthermore, you were right about UV as shown in following link. This makes sense, because molecular probes wouldn't have expected everyone to replace their UV transilluminators just to use the SYBR safe. "How do I view DNA stained with SYBR® Safe DNA gel stain? DNA stained with SYBR® Safe DNA gel stain can be viewed using a blue light transilluminator such as Invitrogen’s Safe Imager™ instrument, or a standard UV transilluminator. If you plan to use the DNA for cloning, avoid exposing DNA stained with SYBR® Safe DNA gel stain to UV light."
- Andy Maloney 08:50, 5 February 2010 (EST): Oh wait. No. It was late and my brain wasn't working. No matter what if we use the Sybr Safe stuff, it will always excite the Cy3 because the emission of the Sybr Safe overlaps with the excitation of the Cy3. Also, I'm pretty sure that the peak in the UV for the Sybr Safe dye is an absorption peak and not an excitation peak.
