You don't want too long of an outgrowth or you'll get replicates of transformed cells.

What happens if you transform with a different plasmid (got any pUC18?)? Do the cells grow when spread on non-selective media? Have you had success with previous transformation protocols?

Got to rule out the obvious before you start contemplating odd scenarios.

Hi Danesh,

in adition to what Katie A S Burnette already recommended I wanted to add some points. It is a very good advice to always include a positive control to see if your cells are viable and if your protocol itself is working and to avoid prolonged post-transformation recovery times.

However, as you working with low copy plasmid you might get colonies with high copy plasmid, but you can still have problems with your low copy plasmid.

I would recommend the following (after verifiying that your are able to transform a high copy plasmid like pPUC 18 - see Katies recommendation):

1. If possible use commercially available competent cells - usually the transformation efficiency is much higher than for competent cells you made yourself.

2. Reduce the concentration of your antibiotic as you have lower amounts of plasmid and therefore the cells produce less chloramphenicol acetyltransferase (which is inactivating the chloramphenicol)

3. Try lower groth temperature e.g. 30 °C for a longer time : 20 h recommended for the pBAD33 vector

4. Try electroporation

5. Make sure that you have really clean, high quality DNA

DEar Danesh Khazaei

generally 1h of growth in a media w/o antibiotic is enough. Longer time in the absence of antibiotic may induce an outgrowtn of empty cells.

Just some questions:

did you test the competence level of your cells with puc18?

Are those cells chemically competent? electrocompetent?

Which amount of plasmid did you used for trasformation?

Was it an empty pBAD33 or containing a GOI?

Which amount of Chloroamphenicol was in your plates?

best

Manuele

Manuele Martinelli Dörthe Andrea Kesper Katie A S Burnette

Thank you for your responses. These cells are chemically competent NEB5α high-efficiency cells. My transformation is successful with a pUC19 control (AmpR//Amp plates) under identical transformation conditions. Unfortunately, my lab does not have a transformed cell stock with a plasmid that confers chloramphenicol (cm) resistance, so I cannot test if the issue lies with the antibiotic stock (whipped up fresh by me a few days earlier). I've plated on different [cm] plates: 10 ug/mL, 25 ug/mL, 33 ug/mL, to no luck. I received this plasmid from our collaborator on filter paper and extracted it with TE. The nanodrop reading is quite suspicious for the amount they say they have provided, with a 260/280 = 1.15. I suspect the problem now lies with the plasmid/DNA quality rather than my transformation. Looks like my PI will be asking for the plasmid to be resent on a different medium.

Hi Danesh Khazaei,

If you've successfully transformed other plasmids with these cells and protocol, and are using standard chloramphenicol concentrations (10 µg/µL should be sufficient), then the problem is likely elsewhere. The most probable causes are either an incorrect plasmid from your collaborator or resistance to a different antibiotic. Don't worry about 260/280 ratios, as they are always bad from filter paper.

You could test antibiotic resistance by growing colonies on plates with other standard antibiotics (e.g., ampicillin, kanamycin). However, you should definitely re-request the plasmid from your collaborator.

I have used pBAD33 quite a few times and generally use a concentration of 30 ug/ml without problem. I often only outgrow for 45 minutes, so I don't think the outgrowth is the problem. Generally with insufficient outgrowth you don't have an abrupt transition from none to colonies, it is more a gradient that will longer outgrowth you get more colonies but you probably would get some without any outgrowth time at all.

I agree the most likely problem is that you don't have plasmid DNA. However you might wish to confirm that your chloramphenicol stocks are good, sometimes a simple math error leads to 10-fold higher concentrations in the stock or the plates.

Ah, this is a classic example of, "Never trust, always verify" when it comes to plasmids

Dried onto a piece of filter paper is the standard way to ship a plasmid. Soak in TE and briefly spin in a centrifuge.

Good luck!