Double Digest, Ligation Problems

[mike60984]

Hello All,

I am attempting to perform a double digest of a plasmid and subsequent ligation reaction. I do the digest, perform the ligation and streak a plate. Then next day there are many colonies present. When I isolate the DNA, however, it is not running at a higher molecular weight on a gel when compared to the vector alone, leading me to believe that the insert was not ligated in. Has anyone else had a problem like this? If so, how did you solve it? Thanks very much.

Mike

[lleyton]

Hi mike,
Have u digest your vector for enough time? If not, the colony presented on the plate might just be a empty one, thus making it weighing bigger.
However, u may double digest ur plasmid to see if the insert is in the vector.
PS: sometimes the correct plasmid may represent wired weight in DNA-GEL due to their different concentration in my personal opinion

[mike60984]

Hi lleyton,

Thank you for your input, I ended up getting it to work!

all the best,

Mike

[kennethmiller12]

Thanks for sharing. As vector is the causing agent of diseases so controlling it disease can be cure.

[Fruits]

Dear Mike,

I was just reading your post. May I know how did you manage to get it to work? I am new here. I did a ligation and transform two different ratios of ligated products (insert: vector ratio), however I got colonies in one plate only. I inoculated the colonies but it seems like the bacteria did not grow at all.

I do not think that they are satellite colonies as I have ampicillin in the agar plate and that I did not incubate for too long a time. So I wonder if anyone has an explanation or encounter the same problem before?

Thank you.

[Warthaug]

A couple of points:
1) Depending on the size of your insert, the vector may not appear to run at a higher mass even if your ligation is successful. Supercoiling, etc, can act to "hide" the true size of larger DNA pieces. Two better ways:
a) Digest the isolated vector with the same enzymes you used for the first digest, and then run the gel. You'll see your insert, at the expected size, if you were successful
b) PCR amplify your insert, using the purified vectors as template. This is my preferred method - I use primers which flank the MCS, so you always get a product. No insert gives a band the size of the MCS, successful insertion gives a product larger. This system is very sensitive, allowing you to find even small inserts. Better yet; you can preform the PCR directly on the colonies on your plate; no need to prep DNA from multiple colonies. Protocols available [Only registered users see links. ], [Only registered users see links. ] and [Only registered users see links. ].

2) To confirm things are working I always ligate and transform the vector without the insert. Assuming that the ends are not compatible, this should produce few/no colonies, while the insert-containing ligation should produce some. If you end up with a lot of colonies in the vector-only plate, either your digest didn't work, your ligation was non-specific, or you are using enzymes which produce compatible sticky ends.

Hope that helps.

Bryan