Help with Cloning

Austin P. So (Hae Jin) nobody at
Wed Sep 29 12:47:51 EST 2004

Prema wrote:
> I am doing a simple directional cloning using KpnI and Not1. What I am
> trying to do is to subclone a TA cloned cDNA into a doubel T7 vector.
> I cut the vector and the insert with these enzymes, run them on a gel,
> cut out the desired vector and insert bands purify through a qiagen
> column and then set up my ligations.

Some clarification is needed...though a picture is worth a thousand 
words, no?

1. how big is the insert relative to the vector?
2. how much are you digesting?
3. how much of your digest are you loading onto your agarose gel?

> When I test the identity of the
> colonies that I get using restriction enzymes, all the colonies test
> positive for one or both of the parent plasmids that I used to get my
> vector and insert fragments.

4. how do you test the identities?

> How is that possible when I cut out the
> right bands from a gel and then ligate them. I usually do my digests
> for 2 hrs and let the gel run long enough before I cut the fragments
> out. Can you give me some suggestions. Also the kpnI and NotI sites
> are in the MCS.

Lots of things can cause this. If you have too much construct, then you 
may be getting incomplete digestion. On a related note, and I don't 
recall exactly, but I think that NotI and KpnI are incompatible as far 
as incubation conditions are concerned, so just in case you didn't, 
these should be done separately (or at least carefully planned...low 
salt to high salt, right?). If you load too much on your gel, you can 
get "parachuting" of larger fragments which can contaminate your 

> It is really frustrating because it looks like a
> simple cloning method but is not working for me!!!!

I think you didn't make the proper sacrifices to the molecular biology 

Seriously though, this is not exact science, so this kind of stuff 
happens all the time...


P.S. what I tried to do early on is to get in the habit of "normalizing" 
restriction enzyme activities to "pmol sites per hour", and then figure 
out how much would be needed at a minimum for whatever I would digest. 
It also helps to figure which gives more bang for the'd be 
surprised at how much prices vary amongst suppliers when you do this.

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