Hi Lakshitha,

I would say it is not normal that a plasmid degrades so rapidly under the correct conditions. You mentioned that the plasmid already contains an insert.

Did you get this from a company or from another lab?

If you got this from a company I would ask them what can be the problem.

Even if the vector is not completly intact I would expect that there might be still some intact plasmids so that you could get some transformants.

However sometimes inserts can be toxic for the cells - try to grow the colonies at lower temperature - sometimes this helps.

You can also try electroporation for transformation - in my hands this sometimes worked better with low concentrations of vector (but still I would try to grow the colonies at lower temperatures).

And double check the vector and the antibiotic resistance - I know this is unlikely but sometimes things get mixed up - maybe try if your vector will grow on kanamycin plates in addition to your ampiccilin plates

Hi Dörthe,

Thank you so much for your descriptive details.

Yes, I ordered them (plasmids with my gene of interest + tags) from a well-reputed company. And as you said, I also thought that there might be a small amount of plasmid vectors left, even though they seemed to be degraded on the agarose gel. So, what I did next was that I transformed 1 uL from the existing plamids (33 ng/uL) into DH5alpha - just to propagate the plasmids. I did this step just to see the possibility of cell growth and the competency of the DH5alpha that I prepared in-house. Then I grew them on an antibiotic-treated (ampicillin) agar medium. After overnight incubation at 37 C, I could obtain very few single isolated colonies (see the attached file). How do you feel about these colonies? Do you think they are really transformed colonies? Now I hope to continue further with these colonies for making pure cultures.

Thanks again for the tips to grow the colonies under low temperatures. This is worth trying from my end.

Since my vector is a pET21a+ plasmid, it has ampicillin resistance. But I can try with your hypothesis in terms of growing them in medium treated with kanamycin.

Thanks

Hi Lakshita,

as you have ordered the plasmid from a company it is likely that you got the correct plasmid and the colonies will most likely not grow on kanamycin plates (however I would try it anyway - nothing to loose).

The number of colonies is very low - this should not happen - would definitvly try the lower temperature as 3 of the look also very small what might be due to toxicity (it will take longer to grow). I would try 30, 25 and 20°C. And I would also inoculated the colonies you have got and do a DNA miniprep - maybe you are lucky.

If nothing work I would nevertheless contacting the company and explain the problem. Maybe they have another idea or they might be generous and replace your vector as it seems that you followed the protocols (you have nothing to loose in asking).

Could there have been a problem during shipping or during storage? I would also look into this - this will most likely help you now but maybe will help avoiding future problems.

I hope it helps an which you good luck!

Hi Dörthe,

Thank you very much for your suggestions. I tried growing the cells under very low temperatures, like you said, but unfortunately, none of them gave me any good results. Since I didn't have anything else to try out, I contacted the company and am waiting for their replies. I hope they will give an opinion on this matter. Truly, this is the first time that I faced such a huge obstacle in transformation.

Hi Lakshitha,

I really hope the company can help you, because these results are really not normal.

Wish you the best