Login

Join for Free!
119257 members


SYBR gold in acrylamide gel

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

Moderator: BioTeam

SYBR gold in acrylamide gel

Postby Kiraya » Wed Oct 29, 2008 9:46 pm

Hello. Someone use de SYBR gold with acrylamide gel? I have a problem, like is written in the protocol I put the SYBR gold on the gel after the electrophoresis in dark and I leave it for 45min but I can't see de DNA (microsatellites). The DNA is not the problem because I can see it in agarose gel. Some one know if there is anything that could interfere with the gel?
Thank you.
Kiraya
Garter
Garter
 
Posts: 4
Joined: Wed Oct 29, 2008 9:31 pm

Postby blcr11 » Thu Oct 30, 2008 1:53 pm

You have anything in your gel or buffer system that quenches fluorescence? Ethidium bromide, maybe? I don't run SYBR gels, so I don't know if that sort of thing happens or not. Another possibilty, I suppose, is that your sample got stuck in the well, but if there is no quenching, I would guess you would have seen your wells light up. I assume you've done it more than once, so there is no possibility that it's just something stupid, like forgetting to put any DNA or reagent in the system.
blcr11
Viper
Viper
 
Posts: 672
Joined: Fri Mar 30, 2007 4:23 am

Postby canalon » Thu Oct 30, 2008 8:01 pm

Let me see if everything is correct:
Your prepare your gel, put DNA and run it, probably to see minor length difference. Tehn take your gel out and stain it for 45 minutes (I usually wait more, but I am working with very minute quantities of DNA). Then put on your UV transilluminator, and using the appropriate filter, try to see your DNA but there is nothing visible.

I guess it would be hard to forget to add the DNA, but there is a chance that it goes out of the gel. Other possibilities: using glassware for the staining step (SYBR gold seems to attach to glass); problem with your SYBR gold (degraded because not stored properly) or wrong dilution in the staining bath; use of EtBr filter instead of the correct filter (should be yellow, not red).

That is all I can see that can go wrong, but I can really forget something.

Good luck and keep us in touch if you find an answer I might be interested.
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)
User avatar
canalon
Inland Taipan
Inland Taipan
 
Posts: 3909
Joined: Thu Feb 03, 2005 2:46 pm
Location: Canada


Re: SYBR gold in acrylamide gel

Postby Kiraya » Fri Oct 31, 2008 9:15 am

Thanks for the answers.
Is the fisrt time that I use SYBR gold. When is finished the run I separated one of the glasses and the gel remains attach in the other glass where I put the bind silane. Then I cover the gel with 1X SYBR gold and I wait 45min-1h on the dark.
I try to see it in the blu UV transilluminator. What do you mean when you say the correct filtre?
I think that the DNA doesn't go out of the gel because the xylene cyanol was in the run and it run more or less at 105bp on 6% acrylamide gel.
May be I should not use the bind-silano?
Thank you.
Kiraya
Garter
Garter
 
Posts: 4
Joined: Wed Oct 29, 2008 9:31 pm

Postby canalon » Sat Nov 01, 2008 3:12 am

I stain in a bath rather than on the glass plate, because according to invitrogen Sybr dyes stick to the glass, but I have not tried to stain your way, so it is hard to know what would happen.

UV or blue light should both work as far as I know and I guess you are looking the gel through a yellow/orange filter. Because many gel documentation system are equiped for EtBr and the dark red filter is far from optimal for the visualization of Sybr gold fluorescence.

Since you see the xylene cyanol, that means that the DNA is correctly loaded. Do you put a molecular weight ladder. If you see it then the problem is likely your sample. If you don't I wonder if your SYBR gold has not degraded...
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)
User avatar
canalon
Inland Taipan
Inland Taipan
 
Posts: 3909
Joined: Thu Feb 03, 2005 2:46 pm
Location: Canada


Return to Molecular Biology

Who is online

Users browsing this forum: No registered users and 2 guests