I am trying to sequence alleles by ligating short PCR products into a pJET 1.2 vector. Although pJET protocol states that the plasmid contains suicidal genes that are disrupted once ligation is successful. I am always getting very low ligation efficiency , I get 4 clones with an insert out of 24.
I cleaned the PCR products before ligation and used a proofreading DNA polymerase to have blunt ends. My template is 647bp in size and has a GC content of 40% which I don't consider an issue. I modified template concentration following to what the protocol suggests, but still getting low efficiency.
Has anybody faced such issue before or have any suggestion?
Make sure to gel extract (avoid UV) your PCR fragments. Otherwise leftover gunk from the PCR, such as primer dimers, will ligate with the cloning vector and result in “empty” clones.
Other than that, pJET1.2 is pretty reliable and, in most cases, should result in 100% positive clones.
Good Luck!
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