I extracted gDNA, amplified the target regions (554bp, 226bp, 344bp, and 194bp), gel extracted the target bands and purified them. Then I did the ligation (3:1 insert to vector) at 4 C overnight, and then transformation following Promega protocol using JM109 competent cells with 50 ng DNA. After gel purification and ligation I got the target bands clearly on the gel. But after plating and 37 C for ~14 h incubation, I got ligation positive control grown well on the plate while both sample and transformation plates only got the blue dots, and the transformation control even grew into such small blue colonies as shown in the figure. The condensed small colonies may indicate that my agar plate was so wet or the plating density was too high, but I am confused whether the blue-only colonies in the samples and transformation control was due to the fail of ligation or the deteriorated competency of the cells. If the ligation was wrong, as there was no visible white colonies in the sampling plates, why the positive control showed a good result and the bands were observed at the right positions on the gel? If the cells were bad, why they all can grow good colonies?
Hi Yang Xiao,
Since the transformation of your ligation positive control worked, I would say your competent cells are fine. I would guess the problem is either the ligation or even before, at digestion step (In case you used two restriction enzymes and only one of them worked, this could also be the reason of ligation failure). How do you judge your correct ligation band? Nicked vector plasmid would also run higher than the intact supercoiled plasmid, could this be the reason you see the higher band or did you run nicked vector plasmid and ligation both in gel for comparison. Can you share you gel picture; this would help in answering your question better.
Archna Archna Thank you for your respond first! I did not need to enzyme digest my DNA since I used primers to specifically amplify the target region by PCR. Then the PCR products were checked on gel and the target bands were gel extracted and purified. Products from purification were checked on the gel again and then I did ligation overnight. When running gel for the ligated product, I ran the positive control with company provided control insert, which is the similar size as my first target; the negative control with no insertion but ligase; and all my samples. All except negative control showed two bands--one with insertion and the other with only uncut plasmid. In comparison with the positive control and refering to marker, all my insertions were there. (I will attach a picture if I figure out how to convert the format into JPEG.)
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