Protocol Online logo
Top : Forum Archives: : Protein and Proteomics

Extremely lunatic competent cell - transformation (Sep/12/2005 )

Pages: 1 2 Next

So.... dry.gif

you have this cutted vector (AgeI SpeI) and a couple of oligo (15 bp of annealing bp +overhang for the two sites) ligate have colonies on Ampicillin plate....

you pick a grow it in liquid LB + Amp....and nothing grow...

is an entire month that i'm dealing with the problem.. changed plate, changed antibiotic...but is always the same...

Some people have suggestion or bet...? blink.gif



How do you prepare your plates for the bacteria? Did you try to see if your agar medium wasn't too hot when adding Ampi otherwise you may have killed the Ampi and your colonies are just empty, growing on a plate without Ampi. Thus when you pick and grow, you just kill the empty bacteria with ampi in the LB.


I considered that but i leave the LB agar in a water bath set at 48 for 5 hours, and then pour the antibiotic...


Did you try to just perform a PCR on a colony to see what is in the bacteria, I mean, is there your plasmid? Did you try to pick your colony with sterile tips to grow it without ampi (at least for few hours)? What is the plasmid, isn't it one of those old plasmids with an old replication origin that needs chloramphenicol (or something like that)? Did you transform empty plasmid you used for the ligation (before cutting it of course)?


I didn't try PCR. Simply, the one that grows sometimes posses the plasmid, sometimes...more surprise...not.

THe vector is a simple pBS ks+ AMP cassette.

I used max efficnecy cells from invitrogen , and this never gave me a problem before...

I use the protocol suggested by the producer, plate....they grow....pick a colony , sterile tranfer in liquid LB...and the colony most of the time dye ( ehe ).

The thing is sometime the whole portocol work fine... blink.gif


Just 2 weeks ago I had the same problem - after transformation I put 200 ul on plate Amp, 350 ul in 100 ml liquid culture. Than incubate both of them at 37°C; On plate there were the colonies, in liquid - nothing. One person told me, that is is the case when the product of protein, encoded by plasmid is toxic. She adviced me to grow bacteria in liquid at 24°C instead of 37°. And - YESS! that worked.


One of my colleagues had a problem similar to this and it turned out to be very simple.

After troubleshooting every single step in turned out that the old autoclave in our building didn't completely sterilize the LB media and that's why her bacteria wouldn't grow.

This probably isn't the answer to your problem but it may be something to consider. Do other bugs (untransformed) grow well in your incubator/media?

Good Luck



Not very much...i don't check the untrasformed so often know...170 dollars for 1 ml of competent cell!!!
But before the untrasformed never grew......
I didn't hink about toxicity, maybe this could be one problem, but when i grew the same insert in an another vector i hadn't any problem...

Brief description again....

My bacteria grow on the selection plate (Amp) and then dye on the liquid media.

Another version (my collegue): the growht on the liquid LB and then contain no plasmid after the miniprep.



this is a shot in the dark but it can cause a problem...

I assume your petris are pre-packaged sterile and all that

what about your test tubes? if your dishwasher needs its filters cleaned, or if sometimes the lab peon doesn't rinse them properly, you could have some inhibitory detergent residue in the test tubes with the LB broth. this would explain why it only shows up sometimes. most of us take our sterile glassware for granted dry.gif

there are simple pH tests to determine if you have a significant amount of detergent residue in your test tube that might inhibit growth

if you want to try this here are a couple methods: both the BPB and the pH meter test are easy and quick, you could look at a random 30 or 40 tubes in a small amount of time and know if this is your problem

good luck


I grow my sample in prepacked sterile 5 ml plastic tube... ph34r.gif


Pages: 1 2 Next