I am using Escherichia coli DH5alpha as competent cells to insert recombinant plasmid. I use pGEM-T Easy Vector from Promega. To ensure my competent cells are not contaminated, I (as my supervisor suggested) usually use ampicillin and kanamycin as antibiotics. Why should I use those antibiotics? Could I use other antibiotics?
your bacteria need to be resistance against antibiotics you use - so no, you can't just use any antibiotic.
I agree with Feng Lin, using two antibiotics seems a bit much.
Oh sorry for not mentioning about this, I forgot. I used those antibiotics when performing "competent cells preparation". So I added those antibiotics before performing the transformation. Also I added those onto two different plates
I performed this step because I want to make sure that my competent cells are pure E. coli DH5alpha. What do you think? Is this step not necessary?
As was already mentioned: E.coli will only grow in presence of antibiotics if it is resistant (e.g. contains an plasmid conferring amp resistance). Your competent cells should be "empty" and should therefore not be resistant against any antibiotics! Resistance is usually used as a selection method after transformation.
If you culture your untransformed cells in antibiotics a small percentage of them will mutate thus becoming resistant. These cells are useless as they cannot be used later for selection. Discard them and start again by making competent cells without further addition of antibiotics.
Hello,
I was wondering : have you already gotten alive cells out of this process? Has your supervisor? If so, was it with the same DH5alpha or with some other already-plasmid-containing cells? (like protein expression BL21Rosetta for which you use chloramphenicol). You should discuss with your supervisor about the detailed protocol.
The DH5alpha strain are modified in a genomic - thus very stable - way, so as long as the person responsible for the strains stocks worked properly (in a sterile way), you should get only DH5alpha in the beginning. Thus as long as some exogenous microorganism doesn't contaminate your plates, you will keep pure DH5alpha cells.
Yes, DH5 alpha carries no resistance to antibiotics. It means it should be manipulated very carefully, using stringent asseptic conditions.
Using two antibiotics helps with selection, making it almost impossible that contaminants grow on the plate. But they require that your cells are resistant to both.
Generally, plasmids come with ampicillin resistance, but you can check that when you order/buy them. Rosetta cells have chloramphenicol resistance, so if you transform Rosette cells with an ampicillin-resistant plasmid, the resulting cells should be resistant to both and both antibiotics can be used in media and plates.
To my knowledge the only purpose of having resistance to two antibiotics is to have selection at two levels, i.e in media and in host. For this, one resistance gene needs to be on the main plasmid body outside of MCS (multiple cloning site) the other one inside the MCS, alongwith gene of interest. After transformation, the resistance gene next to our insert (inside MCS) will be expressed in host being transformed, enabling us to select on transformed host.
Like previous answers, the purpose of using selection antibiotics is to make sure whatever grows in your agar will only be the competent cells that was succesfully transformed with your plasmid. In my case, I even use 3 different antibiotics because I co-transform my bacteria with 2 different plasmid.
So for example, in my case I use BL21 (which is genetically engineered competent cells better at producing protein, because I want to purify recombinant proteins). BL21 has resistance to Chloramphenicol.
My plasmid contains gene that carries resistency to Kanamycin.
My other plasmid contains gene that carries resistency to Spectinomycin.
If you use the original DH5a, it should not have resistency to any antibiotic, so I assume your supervisor asked you to use Amp/Kan because of your plasmid. But usually people will just choose either one, not both.
Why would you use antibiotics, work in Laminar hood near flame and keep your cells on ice bath with flaking ice. Competent cells (dont remember about DH5alpha if it was nalidixic acid that they are resistant to or tetracycline) are way way never to be used with antibiotics as someone already said above. BlUE white screening would require LAIX (LB+AMp+IPTG +Xgal) plates (see promega protocol for pgemteasy T/A cloning manual for screening) clearly mentioned.
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