Hi
I had did not have any yield with mini & Midi prep in last few trials. I used pCDNA3 vectors, so I used Ampicilin as the selective antibiotic in bacteria culture. I got results one time when I used Kanamycin resistance plasmids and Kanamycin as selective antibiotic.
I use chemically competent DH5alpha cells.
So I doubted the problem is Ampicilin and did a control test by spreading transformed (with Ampicilin resistant plasmid) and non transformed compitent cells on Agar Plates with 100ug/ml Ampicilin. Both plates (transformed and non-transformed) had colonies.
So I got new Ampicilin from two other labs and repeated the control experiment. Even with the Ampicilin from other labs, the non transfomed cells gave colonies.
Furthermore, when I used competent cells burrowed from another lab, the control (non transformed cells) did not grow on ampicilin plates while transformed cells did grow.
So it looks like my competent cells are contaminated. I made the compitent cells about year ago and 50ul aliquots were frozen and stored in 1.5ml Eppendorf tubes. Initially, I made 80 vials and I used more than 50 without any problem. I have around 30 vials remaining and now I have above problem. Since the competent cells are aliquotted and as I use one aliquot only once, the remaining stock of competent cells have no chance of contamination.
Theretofore I'm bit suspicious about my conclusion that the competent cells are contaminated. Can anyone provide me some advice or share a similar experience
Thank You :-)
then it certainly is an interesting problem.
I think it's unlikely that your stocks are contaminated. Still, have you tested another vial than the one you actually used for transformation?
Hi Sophie, thank you so much for the answer :-).
I repeated the test with different vials. ( I use a DH5a vial only one time). And I tried with freshly prepared Ampicilin solutions too.
Yes, I too think stock contamination is unlikely. However, the results point towards that direction.
Did you change your ampicillin stock?
Or did you use a lot more cells for testing than you normally use on an untransformed control plate?
It's really weird, but strange things tend to happen.. guess it would be best to throw away your remaining vials and to prepare and test new competent cells.
Hi & Thank you Sophie
I'm sorry I did not see your reply. yes I did stop using my remaining competent stocks as you said (they were 13 months old). And I made new stocks. The new cells are working fine.
Things were really weird. I repeated the experiments three times. Anyway, thank you so much for your advice
Good Luck :-)
Oh My god! Dr Tharaka, We are having the same problem in our lab!!!
In fact, our cells are Top10 OneShot Invitrogen aliquots, we are using these aliquots to prepare new chemocompetent cells because they are old (about 1.5 year) and we thought they were losing competence (because we had a lot of negative colonies growing when doing plasmid constructions).
We tested our new chemocompetent untransformed cells spreading them in LBampicillin (100ug/ml) and LBampicillinless plates. Growth saturated plate without ampicillin (as expected), but still had colonies (aprox 50) in plate WITH Ampicillin.
Then we changed ampicillin stock preparing new plates: Same result.
Then we changed ampicillin flask, new stock, new plates: Same result.
We thought could be contamination of cells, so we took a new frozen cells aliquot (kept at -80°C, having use 1 aliquot per time), grew it, and prepared chemocompetent cells. To be sure, we spread UNtransformed cells in LBamp100 and LB without amp. SAME RESULT. All the plate without amp was covered by cells (again, as expected), but we already had aprox 50 colonies in the ampicillin plates!!!!!
Finally, we prepared new stock of plates with ampicillin (again) and passed 20 colonies of that grown, AND THEY GREW AGAIN.
So I came, to look for someone with the same problem in Researchgate and found this conversation. It´s super interesting how our cells have about one and a half year old, and having the same problem.
I hope someone could explain this....
Hi Brenda
Sorry to hear about the issue you have.
I use competent DH5α cells for transformation. For me making new competent cells from our main DH5α (in glycerol at -80C) stocks worked. Please note that I made new competent cells from our main DH5α stocks, not from the existing competent cells.
If I have not mentioned before, I tried to midi prep the colonies I got as false negatives. I grew them in 4ml LB liquid media with 150ug/ml Ampicillin overnight at 37C in a shaking incubator. After the incubation, the media looked light reddish in color (usually DH5α in LB looks like yellowish brown clouds) and I had a totally different odor compared to regular odor made by DH5α cells (yes, I have a bad habit of smelling some regents I use). Furthermore, the pellet I got after centrifugation was also bit reddish in color and those pellets did not yield any plasmids. The DH5α grown in LB media usually make pellets with yellowish brown color after centrifugation.
The way I found that the issue I had was cells by borrowing a competent cell vial from our next door neighbor lab. That one worked without any issue.
Did you check the false positive colonies you got can yield any plasmids from a mini prep?
If possible try growing those non transformed cells in another antibiotic like Kanamycin.
Something happened during the storage for our cells. That stocks had about 100 vials and I used about 50 of them without any issue. The -80C freezer we kept those cells is a new one and we never had any power failures too. I think the fast way out for you is if you can just borrow a new competent cells vial from a friend. Try those cells and perhaps make new stocks from those cells if they work well.
This whole thing happened to me few years back and I might have forgot some slight details.
Hope this information helps and somebody can provide you with more information.
Good luck :)
Hello Tharaka! Really nice to hear about you.
I think we will do it (borrowing cells) to solve our problem quickly.
But I still have curiosity of what is really happening with our cells.
Also, do not worry about being judged for smelling cells, all the people in our lab do it too, it is a good (ok, a little primitive but still works) way to be sure about what is growing in media.
Well, I think we´ll try doing a miniprep to look for any plasmid.
And I´ll pass the plate to other lab so they can do an antibiogram of the grown colony.
I´ll let you know if something interesting happens.
Greetings for you and your lab!
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