In TA cloning, we have a vector with overhanging T at the ends. so self ligation is not possible. but still we are getting colonies with blue colour. it means there is a gene transcription and which in turn means, vector has self ligated.
Vector indeed self ligate anyway by bacterial recombination. Also, if your insert is small and preserves in frame insertion, you still can see positive blue colonies, but that is quite rare. Do not worry about blue colonies, they should be around 10% of the plate anyway. You would still grow them when performing a control ligation.
Best
francesco
Dear Mr. Gobbo,
My competent cells are indeed recA- and insert size is 1.2 kb. So there is neither chance of recombination nor frame preservation with roughly extra 400 amino acids. still I get blue colonies. what else can contribute?
Thanks.
RecA mostly promotes recombination in closed plasmids. However, there are a number of other DNA-modifying enzymes that reclose open plasmids once they are taken up. I routinely transform RecA- STBL3 strains and they can reclose empty plasmids. If you are curious about it, try ligating the empty vector without your insert, and you will have fewer, but still some colonies, some blue and other white.
If you get white clones from your target ligation, that is fine, and just pick them up.
If you have blue colonies then the lacZ gene is not disrupted and being expressed in the cell. But if the lacZ gene is disrupted then the fragment of interest is inserted in the MCS region of the near the lacz gene and the gene will not be expressed resulting to white colonies, if you are using the pGEM T easy vector kit. Wish you luck!
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