sybr safe

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cmdobson
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sybr safe

Has anyone made the leap from EtBr to sybr safe or sybr green? From my reading it appears the recommendation is to develop the gel for 1 hr in sybrgreen after running. The problem is that people in our lab are used to Etbr so they don't want to wait 1 hour.... Any suggestions? recommendations?

labrat
labrat's picture
Is it possible to run the

Is it possible to run the gels with it in in the first place?

cmdobson
cmdobson's picture
This is what I am not sure

This is what I am not sure about - do you know the concentration required to put into the gel?

Thanks for any information you have

Tony Rook
Tony Rook's picture
As another alternative,

As another alternative, Cambrex (Rockland, ME) recently came out with a hand-held system called FlashGel System. The gels come as cassettes that contain precast prestained agarose gel and buffer. The unit is a combination electrophoresis and transilluminator that is visible under normal lab lighting conditions. Seperation is viewed without UV light and can be observed in real time as the seperation is taking place. Best of all, the gels are complete within 5 minutes. Cambrex is currently running a special where they are giving away the $400 Flashgel Docking system, so all you have to do is buy the gel cassettes. I've already ordered mine but they are currently on backorder. I just thought you might be interested.

Tony Rook

cmdobson
cmdobson's picture
Thanks for that

Thanks for that

Pharmalance
Pharmalance's picture
cmdobson wrote:Has anyone

cmdobson wrote:

Has anyone made the leap from EtBr to sybr safe or sybr green? From my reading it appears the recommendation is to develop the gel for 1 hr in sybrgreen after running. The problem is that people in our lab are used to Etbr so they don't want to wait 1 hour.... Any suggestions? recommendations?

why don't you just run a real-time PCR with SYBR green, if you really want to use SYBR green?

labrat
labrat's picture
Real time PCR doesn't allow

Real time PCR doesn't allow you to
a) look for nonspecific bands
b) cut out your bands for cloning
and countless other things.

I don't think the OP was necessarily saying they definitely want to use Sybr green, just that avoiding EtBr has to be a good thing!

I did have a look at the flashgel system but it looks incredibly expensive for each gel as compared to a bit of agarose.

Any one have any experience with this Sybr green alternative?

cmdobson
cmdobson's picture
That is right - the intention

That is right - the intention is to not use EtBR and use the "safer" version. So we want to use Sybr-safe or some equivalent, but hopefully not pay alot more per gel. Has anyone made the transition from EtBr to syber-safe - what are the results? Can you use sybe-safe in the same way as ETbr - meaning can you add syber-sfe to the gel as it polymerizes?

SRKress
SRKress's picture
cmdobson wrote:Has anyone

cmdobson wrote:

Has anyone made the leap from EtBr to sybr safe or sybr green? From my reading it appears the recommendation is to develop the gel for 1 hr in sybrgreen after running. The problem is that people in our lab are used to Etbr so they don't want to wait 1 hour.... Any suggestions? recommendations?

You easily can add SYBR Safe to the gel as you do when using EtBr (you can even add it before microwafving). The recommended concetration is 1:10.000 (e.g. 3 l to a 30 ml gel). The big disadvantage: You can not reuse the gel the next day since the dye is not stable once it is dissolved in the gel. When you want to reuse the gel you have to stain it after running.

Richard Taylor
Richard Taylor's picture
I've tried a couple of EtBr

I've tried a couple of EtBr replacements and I don't think there's anything that'll beat it in terms of function.

If your problem is disposal of EtBr waste - you can get specially designed filters designed to clean your waste running buffer in the lab - letting you throw what comes out down the drain. It's a charcoal filter - you have to be careful to change it when it's full though.

Schleicher and Schuell filter kit

Leprevost
Leprevost's picture
What is the difference

What is the difference between SYBR Green and SYBR Safe ?

Roshan
Roshan's picture
Leprevost wrote:What is the

Leprevost wrote:

What is the difference between SYBR Green and SYBR Safe ?

SYBR Green and SYBR safe are wonderful products from
Invitrogen, click on the Invitrogen banner under sponsors to get more information:

SYBR Photographic Filter
To achieve optimal sensitivity using Polaroid 667 black-and-white print film and UV illumination, DNA or RNA gels stained with our proprietary SYBR Green I, SYBR Green II or SYBR Gold nucleic acid gel stains (Section 8.4) should be photographed through the SYBR photographic filter (S7569, Photographic Filters for Fluorescent DyeStained Gels and Blots, Figure 23.61). The SYBR photographic filter is also recommended for photographing Southern blots or dot blots stained with our SYBR DX DNA blot stain (S7550, Section 8.5).

SYBR Safe Photographic Filter
The SYBR Safe photographic filter (S37100, Photographic Filters for Fluorescent DyeStained Gels and Blots) is ideal for black-and-white photography of gels stained with the SYBR Safe DNA gel stain (Section 8.4), the safer ethidium bromide alternative. Note that the SYBR Safe photographic filter is identical to the SYPRO photographic filter, which is described below (Figure 23.62).

SYPRO Photographic Filter
To achieve optimal sensitivity using Polaroid 667 black-and-white print film and UV illumination, protein gels or blots stained with any of our proprietary SYPRO protein stains (including the SYPRO Orange, SYPRO Red, SYPRO Tangerine and SYPRO Ruby protein gel stains and the SYPRO Ruby and SYPRO Rose Plus protein blot stains) should be photographed through our SYPRO photographic filter (S6656, Photographic Filters for Fluorescent DyeStained Gels and Blots, Figure 23.62). The SYPRO photographic filter is also ideal for photographing DDAO, used in some of our Western Blot Stain Kits, Glycoprotein Stain Kits and Oligohistidine Blot Stain Kits, which are described in Section 9.4. This filter can also be used with our Pro-Q Sapphire oligohistidine gel stains, which are described in Section 9.4.

helicaldance
helicaldance's picture
We've replaced EtBr

We've replaced EtBr completely in our lab with SYBR I to reduce the level of possible exposure to the proposed carcinogen. We run a dilution of 1/1000 directly with our loading dye and get great results, although I've heard of 1/100 working as well....seems a little skimpy, but to each his/her own.

Direct loading saves the time of staining the gel and has roughly equivalent if not better results for semi-quantitation to EtBr without the level of toxicity.

We also use the SB (sodium borate) electrophoresis buffer which dramatically aids in running gels faster and cooler with decreased "cloudiness" when 1% gels are used.

Using a filter specific for syber will also help with the sensitivity of detection using this dye.

Everybody has a preference, but this is what we use....

hD

bjornson
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I use Sybr Safe and love it.

I use Sybr Safe and love it. I only stain for 30 minutes and don't destain, which is faster than having to stain 15 mins and destain 30 mins with EtBr. Though I know some people don't destain. Bands are easy to see without a filter. I've played with a few filters (not the Invitrogen brand one specifically for Sybr Safe) and they don't enhance the gel that much.

I've stained post-electrophoresis for 30 mins. I know you can add the stain directly to the agarose before microwaving, but haven't tried it yet. I know someone else who loads it with their samples, but I don't know how well that works for small amounts as the dye migrates opposite the DNA (like EtBr). Clare Chemical makes a gel head gadget that allows you to watch the DNA migrate with Sybr Safe in real time, similar to the one mentioned by someone else above but without the 5 minute runtime.

Cheers!

ant
ant's picture
GelStar (1.10000) directly in

GelStar (1.10000) directly in agarose + Bionic Buffer (SB) Sigma
+ Blue Transilluminator ( darkreader, safeimager)

10 min run

SybrGold works fine ( 1:20000)

(do not boil)

SybrSafe claimed resist boiling

cmdobson
cmdobson's picture
Thanks to all for your

Thanks to all for your posting

jagadeesh18
jagadeesh18's picture
cmdobson wrote:Has anyone

cmdobson wrote:

Has anyone made the leap from EtBr to sybr safe or sybr green? From my reading it appears the recommendation is to develop the gel for 1 hr in sybrgreen after running. The problem is that people in our lab are used to Etbr so they don't want to wait 1 hour.... Any suggestions? recommendations?

I add syber safe into agarose solution just before pouring the gel and I use 3UL for every 50 mls agarose.
( conc. of sybersafe is 10,000 X )
It works very fine.

fleck
fleck's picture
SYBR safe is really safer

SYBR safe is really safer than EtBR? I'm worried about using EtBr because I'm pregnant. According to safety meeting, all they can recommend is to handle EtBr when it is added to hot agarose gel under fume hood. The rest of risks of EtBr will be prevented by nitrile gloves and following protocol. I want to suggest that I want to use the alternative, sybr safe instead of EtBr if sybr safe is really safer to the fetus. And, is it helpful of even handling sybr safe under fume hood?

asjenkins
asjenkins's picture
Has anyone used sybr safe

Has anyone used sybr safe with a DGGE gel yet? If so what is the best method of staining...adding to the gel or soaking after running? Thanks for your help!

Katykt12
Katykt12's picture
Can you use Syber Safe if you

Can you use Syber Safe if you plan to clone the PCR product?

Working_Lab
Working_Lab's picture
Hi, I am trying to eliminate

Hi, I am trying to eliminate the use of EtBr and start to look for alternative. I was wondering if we can visualize the gel with Sybersafe on UV light? thanks.

Jason King
Jason King's picture
fleck wrote:SYBR safe is

fleck wrote:

SYBR safe is really safer than EtBR? I'm worried about using EtBr because I'm pregnant. According to safety meeting, all they can recommend is to handle EtBr when it is added to hot agarose gel under fume hood. The rest of risks of EtBr will be prevented by nitrile gloves and following protocol. I want to suggest that I want to use the alternative, sybr safe instead of EtBr if sybr safe is really safer to the fetus. And, is it helpful of even handling sybr safe under fume hood?

I remember a discussion a few years back about EtBr being able to pass through latex gloves. Are nitrile gloves completely safe or is there still a risk?

DKube
DKube's picture
Has anyone tried EZ-Vision,

Has anyone tried EZ-Vision, DNA Dye as Loading Buffer?  Add it to your sample - contains loading/tracking dyes plus fluorescent dye - immediate visualization on transilluminator after electrophoresis.  No staining or de-staining.

philgoetz
philgoetz's picture
No; you use a 470nm blue
philgoetz
philgoetz's picture
Can't afford real-time PCR.

Can't afford real-time PCR.

Andsleet
Andsleet's picture
Cybr green and Cybr safe both

Cybr green and Cybr safe both stain the DNA the same way as EtBr.  As such, there should not be a lot of difference between their mutagenicities, regardless of what the manufacturers claim.
Cybr stains may degrade faster than EtBr, and therefore are a little safer in the environment in the long run.
But no one should add DNA stains to hot gels, period...... a very bad practice by a lot of students.
When one runs electrophoresis gel with EtBr already mixed in the gel, the EtBr runs out of the gel matrix and contaminates everything....... bad situation.

Biju
Biju's picture
Hi

Hi
I have seen a system similar to flash gel; but it is not a  hand held sytem.But you could see the DNA band by turning on a switch when the DNA will be illuminated by sybrSafe(?I am not sure).The problems are 
1.Yyou cannot use the band  for Sequencing applications by fluorescent method
2. If you fail to collect the DNA at a particular time(that  should be optimized), you will not be able to retrieve the DNA.
Hope this helps.
Biju