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Blue Juice loading dye & Gel extraction kit - HELP :( (Aug/05/2009 )

Hi everyone

I just ran a gel of my PCR amplified oligos today and for that gel, I used 10 times Blue juice( 1part blue juice with 4 parts DNA).
Then when I tempted to extract the DNA from the agarose with QIAGEN gel extraction kit, upon adding the QG Buffer and dissolving the agarose, the colour of the QG was a strange blue-green which I had never seen before. I was wondering why the colour was not orange like it is suppoesd to be?? Also in the protocol it says if it turns a violet colour to adjust pH by adding sodium acetate but this is a wierd blue-green colour. Im thinking its the blue juice.....can i still continue with the gel extraction protocol and ignore the colour????What should I do??? i really do not want to restart the whole PCR again.

Thank you so much

-Ms new scientist-

I would continue. Qiagen has a pH indicator added to their QG buffer which changes color if the pH is off. I don't think that's what you're seeing here; it's likely just blue juice getting in the way. So, if the blue juice wasn't there, everything would look normal.

BTW, we switched to an Orange G tracking dye to avoid just such interference by blue juice (which is 0.3% (w/v) bromophenol blue, 65% (w/v) sucrose, 10 mM Tris-HCl (pH 7.5), and 10 mM EDTA). Here's our recipe:

Orange G loading buffer/tracking dye (6x, 100 ml)

0.25 g Orange G (Sigma O3756)
15 g Ficoll 400
2 ml 50x TAE

Bring to final volume of 100 ml with sdH2O.
Store at RT.

Ficoll (15% w/v), glycerol (30% w/v), or sucrose (40% w/v) can be used interchangeably.
Orange G is sometimes used in concentrations up to 0.4% w/v.

-HomeBrew-

I keep my Orange G buffer in the freezer and on ice when in use. While it is said that bacteria will not grow on Ficoll, my lab has found that to be untrue. Given enough time (weeks) of normal use, bacteria will start to grow and this can/will lead to degradation of your sample.

Preparation of 10x Orange G.
1- Add the ingredients to a 250ml bottle

0.25 g Orange G (Sigma O3756)
25 g Ficoll 400
2 ml 50x TAE

2- top up to 100ml.
3- Autoclave the mixture.
4- Aliquot Orange G buffer into 1.5ml micro-centrifuge tubes.

While it is "possible" to get Ficoll into solution by mixing at rt, you will find the process slow, sticky and very messy. It is easier to put all the ingredients into a bottle and autoclave. The ficoll will go into solution without trouble.

Aliquoting the Orange G buffer into microcentrifuge tubes is a must, dried orangeG buffer is a surprisingly good glue and can gum bottles (plastic or glass) shut that nothing but a two day soak in water will get the blasted cap off. (I have accidentally shattered bottles filled with Orange G, while trying to get the cap off - a very messy experience). Popping off a micro centrifuge cap is a much easier experience.

-perneseblue-

Thank you so much everyone for replying and for all the help :)
I am continiung with my gel extraction despite the strange blue colour.

-Ms new scientist-

Ms new scientist on Aug 6 2009, 10:27 AM said:

Thank you so much everyone for replying and for all the help :D
I am continiung with my gel extraction despite the strange blue colour.



Because your DNA migrates at the same speed as your color dye

Next time, use another dye.

Peter
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-dealsvista-

That's interesting, perneseblue... While I don't doubt your experiences, we've never had to keep Orange G at anything but room temperature, have never even considered autoclaving it (though we do use sterile distilled water to make it up), and don't usually bother to aliquot it. No problems going on ten years or so...

While I haven't made it in a while (100 ml lasts quite a long time), I don't recall any difficulty making it, though I'm less certain here...

-HomeBrew-

Hmm.. interesting.

I wonder if the difference is caused by the concentration of Orange G buffer made, 6x verses 10x.

-perneseblue-