I'm trying to revive glycerol stock of E. coli DH5alpha by streaking on a Nalidixic acid (NA) containing plate. I have prepared fresh nalidixic acid (15ug/mL) but I'm getting mixed reviews on the optimal concentration of NA to be used. Also, some suggest making it in NaOH and others in water. If it is NaOH, then what dilution should be used to dissolve? When I streak the cells in NA containing plate, even after 12 hours colonies are too small to inoculate. When inoculated in LB with NA then also the growth is very less after 14 hours. Do you think it is the problem with NA or the cells?
Thanks in advance!
Honestly if it's giving you trouble I wouldn't even bother, just streak it on a plain LB plate. DH5a's nalidixic acid resistance comes from a point mutation in the chromosome, it's not going anywhere even if you don't select for it.
If you're trying to prevent contamination, nalidixic acid isn't going to do much since it only works on certain gram negative organisms. Most media contaminants I've seen are gram positive organisms like Staph species, which have intrinsic resistance to nalidixic acid.
Hello,
Concentration does not depend on Bacterial strain like DH5a. If you are transforming it with some kind of vector, you should check the optimal concentration of antibiotic which your plasmid can tolerate or as recommended by the manufacturer. Give more time to grow.
I hope it will work
Dear Aditya,
Thanks for your suggestion. I do have a selectable marker in the vector and I use the standard concentration of the antibiotic accordingly. I want to select the competent cells first and then the transformants. I need to make sure that they are indeed E. coli DH5alpha, so I need to use Nalidixic acid. Is there time till which Nalidixic acid is supposed to remain active, the maximum time the incubation can be stretched to? I'm concerned because, recently while cloning we got numerous spurious (false positive) colonies and we thought may be there are some contaminants growing along with DH5alpha and no better way to avoid them than using Nalidixic acid, but the DH5alpha themselves are growing slowly there.
Ok I got it. There are few things to check:
1. Spread your competent cells to see whether they are not contaminated.
2. Don't use antibiotic older than a month.
3. False positive....I also get. Actually antibiotic itself start degrading so it gives space to those bacteria which are non-transformed and they start growing. So when you pick up your clones make sure they are bigger in size.
4. 16 hrs incubation is fine I think, no problem.
5. Please check the concentration of antibiotic that your plasmid can tolerance.
Thanks
Hi Aditya,
1. To see whether they are contaminated or not I spread them in Nalidixc acid containing plate, but since they are growing too slow, I'm doubting.
2. My antibiotic is ampicillin and I have used freshly prepared ones only.
3. Earlier I used to get almost all positive colonies, using similar vector:insert ratio (rather, same vector, different inserts), but now I'm getting too many false positives. I know that satellite colonies start appearing nearby when the ampicillin is degraded by the main colony. So I pick the bigger ones which have apparently appeared earlier.
4. I remove the plate after 12 hours as resistant colonies appear by then and they are big enough to inoculate.
5. It is the standard concentration of ampicillin used everywhere and by me as well, all the time and it has worked fine previously.
Thanks again.
Sometimes your Gene of Interest may cause some problem as well. If your main concern is not specifically with DH5a, then please change competent cells. Use some other strain of E.coli. I faced the same problem during Cloning one of my Gene. Finally, I changed my E.coli strain and it worked
I was using E.coli JM109 and trying to clone a gene and every time there were no clones at all while there were some colonies on Only Vector Control Plate. Theoretically, there must be at least same number of colonies on the plate (Vector+Insert) as Vector control plate. So, there was only possibility that my GOI is not compatible with that strain. Then I tried TOP10 and DH5a and finally got the positive clone with DH5a.
So, I will suggest you try other strains and I hope it will work for sure,
Thanks
Dear Aditya,
The genotype of E. coli DH5alpha is: F– endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG purB20 φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK–mK+), λ– . If you see it carefully, it contains gyrA96 mutation which provide Nalidixic Acid resistance. So, Tias is right in using Nalidixic Acid while reviving the cells as it would limit growing of contaminants in the plate.
Dear Tias,
I guess your concentration of Nalidixic Acid is sub-optimal. At lower concentrations, Nalidixic acid acts in a bacteriostatic manner (inhibits growth and reproduction of bacteria) while at optimum concentrations it is bactericidal (kills bacteria). Hence, at sub-optimal concentration you may get minute colonies. I generally prepare Nalidixic Acid in NaOH (0.15 M) and use at 25 ug/mL concentration. I am attaching a laboratory exercise protocol which I follow. Hope it will work for you too. Best wishes.
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