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DNA loading buffer


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#1 amdo

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Posted 17 June 2010 - 06:49 AM

Hi all brothers and sisters;

I intend to use agarose gel electrophoresis next days to see my plasmid but I am wondering about the loading buffer. I do not have xylen cyanol. can I use TE buffer only to load DNA samples. I have already Fast Blast DNA Staining solution from Bio-Rad and it can be used to stain the DNA during the electrophoresis run.

please help..

thanks

Fast Blast DNA Staining solution from Bio-Rad :

((
Fast Blast DNA Stain

Fast Blast DNA stain is an ultrasensitive, convenient, inexpensive, safe, and nontoxic alternative to ethidium bromide for the detection of DNA. This unique product stains DNA deep blue in both agarose and polyacrylamide gels, providing vivid, consistent results. Agarose gels stained with Fast Blast stain can be air-dried on our unique gel support film. Once dried, gels can be kept as a permanent record of the electrophoresis run.

Fast Blast DNA stain is packaged as 100 ml of a 500x concentrate that must be diluted before use. Use Fast Blast stain to:

* Stain DNA in agarose gels after electrophoresis in less than 15 minutes or overnight (protocol in pdf)
* Stain DNA during electrophoresis (protocol in pdf)
* Teach students basic principles of electrophoresis (protocol in pdf)
* Stain nuclei in intact cheek cells (protocol in pdf)

In addition to staining DNA bands in agarose gels, use this stain to teach basic principles of electrophoresis. Fast Blast dye molecules are positively charged, and when placed in an agarose gel, will migrate towards the negative electrode during electrophoresis, providing a striking and inexpensive visual demonstration of the movement of molecules during electrophoresis.
))

http://www3.bio-rad....elStainging.pdf

thanks again

#2 phage434

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Posted 17 June 2010 - 01:25 PM

Loading buffer has two functions. One is to make the solution high density so that it sinks in the well, another is to dye the solution so that you can see it. Pretty much any dye will do. We use orange-g, which moves at the front of the DNA and does not obscure bands as xylene cyanole does. You can raise density with either ficol (which we use at 15% for 6x loading buffer) or sucrose, which you can use at higher concentrations (I think around 40%). Ficol is stable at RT, sucrose may develop growth.

#3 amdo

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Posted 19 June 2010 - 05:57 AM

Loading buffer has two functions. One is to make the solution high density so that it sinks in the well, another is to dye the solution so that you can see it. Pretty much any dye will do. We use orange-g, which moves at the front of the DNA and does not obscure bands as xylene cyanole does. You can raise density with either ficol (which we use at 15% for 6x loading buffer) or sucrose, which you can use at higher concentrations (I think around 40%). Ficol is stable at RT, sucrose may develop growth.


Thanks a lot.
I finally found and borrowed some if loading buffer.. so no problem now.




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