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problems with subcloning - (Dec/20/2008 )

I've been trying to sub-clone a 2.5 kb insert into pEGFP-N1 vector by using REs XhoI and KpnI. I keep getting a 1:1 ratio between number of colonies in the ligation of the insert and vector, comparing to the vector itself (no insert). I can see the vector is cleaved by the REs, as it contains an insert, and when I run the gel I can see both fragments. Can anyone offer some advice? I tried several times but keep getting the same results, and of course the colonies I got do not contain the new insert.


Are you purifying the vector away from the insert you cut out? Are you dephosphorylating the vector?

Small amounts of uncut vector will transform efficiently, and if the insert fails to ligate, your ligation and vector only control will appear identical.

Possible problems: UV exposure of your insert. uncut ends of the insert. failed ligation.

We'll need to hear a lot more detail about what you are doing to offer much more help.


Agree with phage 434.
It can be many things in addition- so please provide a bit more detail of what you have tried etc..


Yes, I am purifying away the vector, and I tried dephosphrylating it.
My gene of interest is a PCR product from a plasmid. I can see a clear band PCR product that has the correct size. I clean the PCR product and cut it with XhoI and KpnI, clean it again and then put it for ligation with the cut plasmid. So the gene of interest is not exposed to UV at any time.
How can I know the insert was cut properly? And how can I tell if the ligation didn't succeed? What else can be the problem?


For the ligation you could do two things
1) for checking if the ligase is fine you can ligate DNA ladder and put check on a gel.
2) Set up two reactions e.g. 20µl each: (for example 20-40ng vector + (x ng) insert + ligase and the second without ligase. load 10-15µl on gel. You can check if the insert is 'gone' in the reaction that contained ligase (or additionally contains products which are not present in your reaction without ligase). I usually perfom this control and it is quite reliable.

Sometimes the restriction doesn't work nicely on pcr products if the restriction site is on the very end of the primer. So I could imagine you have a problem there.
Don't know how many colonies we are talking about but it seems also likely that the dephosphorylation of the vector did not work perfectly since you have the same amount in your religation control - so you should try to get 'zero' (well just a very few!) colonies in your religation control to make sure you have no contamination with religation or uncut plasmid.