After checking my insert and vitality of cells, I made the cells competent but transformation still does not work. I then need to be sure that the cells are competent.
Hello Justin,
therefore you can use plasmid DNA which you can buy and then checking with a definite concentration (10 pg DNA) and then you can see after transformation the competent. If you have competent cells in your lab look into the kit, often there is a control plasmid DNA inside.
Hello Justin,
therefore you can use plasmid DNA which you can buy and then checking with a definite concentration (10 pg DNA) and then you can see after transformation the competent. If you have competent cells in your lab look into the kit, often there is a control plasmid DNA inside.
Use positive control( an other plasmid /construct having same selection marker) . if you are not getting colonies from +ve control it shows that your cells are not competent or lost their competency.
As previously recommended, using a control is the best approach. However, it might be also worth checking ampicillin concentration used in your plates, length of the insert, PCR products purification (if cells do not grow). If they do, but they are all blue, I would check if they have the insert anyway (after extraction and purification run them in a gel).
Good luck!
Just transform a circular plasmid (from a high quality prep). Using 10 pg of plasmid you should obtain 10E9 bacterial colonies per microgram of plasmid for chemically competent bacteria (or higher for electrocompetent cells). But it should be noted that, paradoxically, you cannot obtain such an efficiency if you transform 1 microgram of plasmid. So you shoud have 10 000 colonies for 10 pg of plasmid.
I have used a control plasmid pUC19 on ampicillin plate and i got colonies on positive plates. So cells are still competent.
Thanks to all for contributions.
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