electrophoresis/ difference between expected size and result/ what's the alt - How to confirm the band means the expected DNA? Sequencing&what else? (Aug/04/2010 )

Hi guys,

I got bands from the agarose gel electrophoresis. My expected size was 0.81 kb, but I got 0.67 kb. I cut the blunt vector ligation product (recombinant blunt vector) with a restriction enzyme. 0.81 kb was expected insert.

I need to confirm that 0.67 kb DNA actually means 0.81 kb DNA. What can I do?

I thought about 1) sequencing.

2) Southern blot can be done (but it will show basically the same result as the gel electrophoresis)
3) Hybridization with probe by using DNA chip may be expensive.


What else can be done?
What is the easy way?
I should be able to confirm those are the same DNA.


I NEED TO GET AN ANSWER TONIGHT.
I APPRECIATE FOR YOUR HELP IN ADVANCE.

JA

Eternal city on Thu Aug 5 00:25:37 2010 said:

Please correct me if I am wrong.
You have an insert ligated into a vector. You have cut this plasmid with a restriction enzyme. The expected fragment size is 800bp but you got a fragment which is 670bp

What kind of gel are you running your DNA on? Use a 1.5% agarose gel. Run the gel at the highest voltage possible.

Run your digest again. However this time desalt the digest before running it on the gel. Ethanol precipitate the digest, followed by a 70% Ethanol wash. Dry briefly at 68C> Then resuspend in water and then run sample on gel.

At the same time, you should also cut the plasmid with different restriction enzymes. THese should produce an easily identifiable pattern that would confirm if the 800bp is shorter by 200bp.

Also do not overload your well. Ideally there should be just enough DNA to give band the thickness of a pencil line. (Over load and you get a big fat band.)

If the digest still say that the band is 200bp smaller, I would attempt to isolate a second, third or forth isolate of the desired plasmid. Do the digest on those clone.

You can sequence you clone... but experience say that 200bp is a big enough difference to be real. It is probable that the plasmid is not right.,

perneseblue on Thu Aug 5 03:07:03 2010 said:

I think you are absolutely correct.
As you mentioned, restriction mapping can be done.

I use 0.8% agarose gel. I am going to use 3% NuSieve gel this time.

Thanks a lot.

Eternal city on Thu Aug 5 00:25:37 2010 said:

Which fragments do you expect if your insert is ligated in the reverse orientation in your vector? Since you say it's a blunt end ligation, the insert can be ligated in two orientations. So maybe if you check other clones it will be in the expected orientation.

But anyway, sequencing would be the next option for me. That's the only way to know for certain what you are dealing with.

dpo on Thu Aug 5 14:06:32 2010 said:

I think you are right about the orientation. But I digested the recombinant blunt vector with EcoRI because the vector has two EcoRI site right next to insertion site. I think the size matters in this case, not the orientation.

Thanks for the reply.