So let say I have circular plasmid named "PDR111". I did PCR with KOD ONE MIX KIT and the product is linear DNA. Then, I did self circularization of linear DNA with T4 DNA Ligase protocol (Thermo Scientific #EL0014). I did everything right as the protocol said, But my transformation keep fail. Here's how my electrophoresis looks. My Plasmid size is 11871 kb. Pls guide me
you can use vector sequencing primers to confirm whether your gene has been ligated to the vector. But it is better if you use vector sequencing primers after transformation and check the same with colony PCR.
if your oligo are not phosphorylated you will not be able to self ligate... need phosphorylated oligo or restriction site at the ends of the fragment....
alternatively you can treat the PCR product with T4 polynucleotide kinase + ATP. to control ligation you should see a "scale of DNA or a much larger product on your electrophoresis
Most people don't bother with checking for ligation & just proceed to transformation. Include a full set of controls, including the pUC18 plasmid that comes with most kits. That can help you determine if the issue is with the transformation protocol, the competent cells, or specific to your plasmid.
Good luck!
So you have no insert? Just amplify/linearize your plasmid and then ligate it back?
Tomáš Hluska i dont have any insert, i had done PCR to modify 20 - 30 nucleotides on RBS, and then my supervisor ordered me to do self circularization
Jeremy Mullesa as Didier Poncet wrote, you must have phosphorylated DNA to ligate it. If your primers are not phosphorylated, you can use a kinase to add the phosphate.
Alternatively, if you used primers with overlaps, the bacterium should be able to recombine it by itself.
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