TA cloning: ligation problem or toxic construct? - (Apr/23/2013 )

Dear all,
I tried to ligate my insert (520bp) into TOPO vector (TA cloning).
My PCR product has concentration of 30~40 ng/mcl
I used the mixture as below:

PCR product 3 mcl
TOPO vector 0.3 mcl
Salt 1 mcl
D.W 5.7 mcl

Incubate at RT for 30 min
Than transfer the mixture into Competent cell.
However, I just got the blue colonies, without any white colony. I did try 3 times with the same results.

Previously, I did the same protocol for another construct (625bp), The results were good.
I don't know what could cause the problem for this new construct...
Please give my some suggestions.
Thank you so much!

were you using a kit for ligation? any ligase present? What polimerase used in PCR? Was the PCR product cleaned up (column or gel extraction) before using it and how the concentration of it was calculated/measured?

Clean your PCR product. TOPO shouldn't religate on itself. Sounds like excess ATPs left over from PCR are what's inserting. And be sure you're using Taq polymerase.

There is no ATP in a pcr reaction, only dATP (which perhaps is what you meant). I'd go for the toxic product theory myself, especially if your positive control (your other cloning) works.

Hi all,
Thank you for suggestiones. Here are some more details:
I used EX Taq polymerase for PCR, after that I did gel elution to get the inserts.
According to the protocol of TOPO vector, the ligation does not require any ligase.

And, I could get the blue colonies, but not white colonies.

If the constructs were toxic, should I incubate the agar plate at room temperature? Or is there any way to solve this problem?
Thank you again....