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