I'm trying to intercalate YOYO-1 stain into lambda phage DNA so I can do the kind of visualisation you're talking about. I follow a protocol from Su et al who have DNA 5pM and dye:base pair 1:4 ratio. I mix DNA and dye and incubate in dark at 37 deg for 90 mins following their paper but I see nothing. Seems like intercalation isn't working. Any ideas why it might not be working? What protocol do you use?
You can view my complete protocol in my thesis on Caltech's thesis archive (look for Paul Grayson), but I suspect that your problem has to do with some specifics of what you are doing. What is your filter setup, and have you ever been able to observe YOYO fluorescence?
Good Demonstration here. I do the same DNA stretching with Lambda with the YOYO-1 dye. Though my dye is quickly photobleached (10 seconds) so I am not able to capture a clip as long as yours.
Do you have any experience with electrophoretic stretching of Lambda?
10 seconds is about right for dye with no photobleaching treatment. To get the longer photobleaching time, you should look into Paul Selvin's anti-photobleaching formulation involving glucose oxidase and catalase. See my thesis at the Caltech thesis site (under Paul Grayson) for exact details.
Yup, they sure are. The microscope doesn't actually have to be that amazing to capture single DNA molecules - what you need is extremely bright fluorescent dyes that you can attach to the DNA to make it visible.
Did you read the description? Each one of those lines is the DNA emerging from a single virus particle. The flow of water past the sample is the "wind" that stretches out the DNA to make it visible.
Noooooo now the viruses are just proteins and never got to complete their primary function correctly D:
CuppaS00p 1 year ago
that is cool.
jessiep02 2 years ago
I'm trying to intercalate YOYO-1 stain into lambda phage DNA so I can do the kind of visualisation you're talking about. I follow a protocol from Su et al who have DNA 5pM and dye:base pair 1:4 ratio. I mix DNA and dye and incubate in dark at 37 deg for 90 mins following their paper but I see nothing. Seems like intercalation isn't working. Any ideas why it might not be working? What protocol do you use?
Mike
ppxmis1 2 years ago
Hi Mike,
You can view my complete protocol in my thesis on Caltech's thesis archive (look for Paul Grayson), but I suspect that your problem has to do with some specifics of what you are doing. What is your filter setup, and have you ever been able to observe YOYO fluorescence?
-Paul
pdg137 2 years ago
Good Demonstration here. I do the same DNA stretching with Lambda with the YOYO-1 dye. Though my dye is quickly photobleached (10 seconds) so I am not able to capture a clip as long as yours.
Do you have any experience with electrophoretic stretching of Lambda?
Thanks
Carl
carldale 3 years ago
Hi Carl,
10 seconds is about right for dye with no photobleaching treatment. To get the longer photobleaching time, you should look into Paul Selvin's anti-photobleaching formulation involving glucose oxidase and catalase. See my thesis at the Caltech thesis site (under Paul Grayson) for exact details.
-Paul
pdg137 2 years ago
Good bye lambda! Do this with all virulent viruses as well so we don`t get sick :) But then some unwelcomed bacteria will cope :(
Moshpitssser 3 years ago
It is an spectacular video! I love to see it, Is is like to be in other dimension.
Congratulation to the author!
miranda4538 3 years ago
are the white strips actually the dna molecules?! cuz thhat is a pretty good microscope.
irkiIIer 4 years ago
Yup, they sure are. The microscope doesn't actually have to be that amazing to capture single DNA molecules - what you need is extremely bright fluorescent dyes that you can attach to the DNA to make it visible.
pdg137 3 years ago
This is really cool. Its a great example of physics at a microscopic level.
thepryest 4 years ago
This is really fantastic! What kind of signal do you use to get them to do that?
fbgenius 4 years ago
Bacteriophage lambda automatically ejects its DNA when triggered by its receptor protein LamB, which was added to the solution in this experiment.
pdg137 4 years ago
Excellent demonstration pgd137!
ehlolz 4 years ago
i don't get it.. looks like DNA being stipped off by wind..
kciNJNadon 4 years ago
Did you read the description? Each one of those lines is the DNA emerging from a single virus particle. The flow of water past the sample is the "wind" that stretches out the DNA to make it visible.
pdg137 4 years ago