Hi Guys
I am trying to clone my PCR product into PUC19 with blunt end ligation. I amplified my PCR product using phusion High-fidelity DNA polymerase (NEB). I cut the vector with SmaI and treated it with alkaline phosphatase shrimp (Roche). Both PCR product and dephosphorylated vector were gel purified. I use DNA T4 ligase at 16 degree O/N with insert:vector ratio of 1:5. But I got no colonies. positive and negative control all worked well. Just wondering will PCR product amplified using phusion HIFI DNA polymerase have 5' phosphate groups? Or I should add P to my PCR product?
Thanks
Hi there,
If your primers are not phosphorylated then the PCR product is not phosphorylated and as the digested vector is not phosphorylated it does explain why you don't get anything out of the ligation/transformation reaction. Apart from phosphorylating the PCR product and ligate it with dephosphorylated vector, you may also try an other technique which is valid only if the expected ligation is destroying the SmaI site : you may run the ligation with the non phosphorylated PCR product and the SmaI treated plasmid (without AP treament) in presence of SmaI enzyme (SmaI will redigest any SmaI site reformation whereas expected ligation will become SmaI insensitive).
Whether a PCR product contains 5' phosphates or not depends on the primers, not on the polymerase. Most oligonucleotides are synthesized with a 5' OH (i.e. are dephosphorylated) so most likely your PCR product cannot be ligated to a dephosphorylated vector.
If you have some PNK and ATP, it is easy to phosphorylate the PCR band, though. Or you can phosphorylate the primers beforehand and then do the PCR.
Hope it helps!
Hi there,
If your primers are not phosphorylated then the PCR product is not phosphorylated and as the digested vector is not phosphorylated it does explain why you don't get anything out of the ligation/transformation reaction. Apart from phosphorylating the PCR product and ligate it with dephosphorylated vector, you may also try an other technique which is valid only if the expected ligation is destroying the SmaI site : you may run the ligation with the non phosphorylated PCR product and the SmaI treated plasmid (without AP treament) in presence of SmaI enzyme (SmaI will redigest any SmaI site reformation whereas expected ligation will become SmaI insensitive).
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