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Weird two bands of my purified vector DNA apeared on my gel ? - (Aug/13/2007 )

Hi everyone

I cut my DNA vector with AgeI and AseI , then purified the 4.1kb vector by gel extraction with column. After purifying, I checked DNA on agarose gel.

It should be showed only one band on 4.1kb. How come was the other weird band appeared below 4.1kb???(I don't think "wrong operation" during purification results in weird two band, as I did the same way to purify my insert DNA, and this situation did not happen.)

And...(I am not sure how that inspiration come from my mind), then I checked again my vector DNA with loading dye mixed with 10mM EDTA . It returned to the single sharp band on the right place!!!

Is it possible that during electrophoresis my vector is very easy to denature and adding EDTA could protect it from denaturing?? Or my vector DNA have denatured after purification, and addind EDTA during electrophrosis could help it renature??

After purification, I have to proceed to ligation. But now I am not sure whehter my vector is well or not?? and if it is not well, during which step made it denature?? and why EDTA could change this situation?? ...........

I have so many questions and so confused... Could anyone give me some suggestions?? THANKS A LOT!

[attachment=3428:__22294___29255_2.png]
Left: vector checking with EDTA
Right: vector checking without EDTA

-April0210-

I would proceed with transformation. check some colonies for the right product (by restriction analysis). this will only take one day of work.

then you could check the vector-insert junction by sequencing. (needs longer, but you will surely know if your vector is ok)

-moljul-

Just go ahead for ligation and transfromation.

I think maybe there are some secondary structure on your vector. So it gave you two bands.

Good luck!


QUOTE (April0210 @ Aug 13 2007, 05:25 AM)
Hi everyone

I cut my DNA vector with AgeI and AseI , then purified the 4.1kb vector by gel extraction with column. After purifying, I checked DNA on agarose gel.

It should be showed only one band on 4.1kb. How come was the other weird band appeared below 4.1kb???(I don't think "wrong operation" during purification results in weird two band, as I did the same way to purify my insert DNA, and this situation did not happen.)

And...(I am not sure how that inspiration come from my mind), then I checked again my vector DNA with loading dye mixed with 10mM EDTA . It returned to the single sharp band on the right place!!!

Is it possible that during electrophoresis my vector is very easy to denature and adding EDTA could protect it from denaturing?? Or my vector DNA have denatured after purification, and addind EDTA during electrophrosis could help it renature??

After purification, I have to proceed to ligation. But now I am not sure whehter my vector is well or not?? and if it is not well, during which step made it denature?? and why EDTA could change this situation?? ...........

I have so many questions and so confused... Could anyone give me some suggestions?? THANKS A LOT!

[attachment=3428:__22294___29255_2.png]
Left: vector checking with EDTA
Right: vector checking without EDTA

-spore-

I've seen exactly the same thing when purifying using a column. After a while I figured out that I only see it in loading buffer without tris/EDTA (I first noticed it when I moved labs and foolishly changed recipes).

As I understand it (which was confirmed by the Qiagen tech support), this is as single stranded (i.e. denatured) DNA comes off the column. Hence adding EDTA allows reassociation and your DNA runs as "normal". I've experienced no problem using this DNA in ligations etc.

I wouldn't worry about it - i'd just add EDTA to your loading buffer in future!

-draddoga-

QUOTE (draddoga @ Aug 13 2007, 01:02 PM)
I've seen exactly the same thing when purifying using a column. After a while I figured out that I only see it in loading buffer without tris/EDTA (I first noticed it when I moved labs and foolishly changed recipes).

As I understand it (which was confirmed by the Qiagen tech support), this is as single stranded (i.e. denatured) DNA comes off the column. Hence adding EDTA allows reassociation and your DNA runs as "normal". I've experienced no problem using this DNA in ligations etc.

I wouldn't worry about it - i'd just add EDTA to your loading buffer in future!


SO...you mean.. EDTA makes vector DNA "look like" "normal" on gel or it just have been fine after column purifying??

Could I proceed ligation directly or should I have to add EDTA to my purified product to make vector renature??? (but EDTA inhibits ligase activity, dosen't it??)

by the way....^^ I am really touched by that you've seen the same thing with mine, cuz I've discussed this matter with a lot people. Even my advisor told me it was resulted from "wrong operation" and "reagent contamination" ...@@


thanks for your help... tongue.gif

-April0210-

Your DNA should be fine for ligation - it should renature in the salt in the ligase buffer. I'd just go with what you have!

-draddoga-