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ligation and transformation - (Nov/27/2004 )

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I reverse amplified a 6 kb portion from a 8kb plasmid.
I gel purified the PCR product using Qiagen Gel purification kit and digested it with Nhe1 and set up an overnight ligation which should yield a circularized vector (self ligation).
I then transformed and I used-Km (25ug/ml final concentration) as the marker to select for transformants.
The plates with the negative control (only competent Dh5 alpha) look funny, because I see a lawn of bacteria growing- its weird since they are all over the plate.
My other plates with the transformants also have the same appearance.
I don't think the competent cells are the issue since I purchased ready to use competent cells. I changed the medium containing Lb-Km just to eliminate possible contamination.
does anyone know why I see this lawn? I have never seen anything like this.
i don't think its the plates since I made them fresh and checked the antibiotic concentration.
Please Help!!!!

-researcher-

Did you add antibiotic to hot LB? Or autoclave the LB with amp ?
It sounds like the AMP is degraded. Make a fresh batch and add it to
the LB when it is cool enough to touch to your arm.

-mikew-

Hi
i hope the antibiotic final conc. used should be increased to 60 ug/ml.
may be you can check the transformation again (6 kb).
Do you check for the plasmid by rapid lysis for the so called non-transformants (lawn).
you didn't mension about the +ve control. if you have not used you can also keep an appropriate positive control to check the success of you transformation experiment.

Best regards,
RAVI
INDIA

-ravibiot-

Thank you for the replies.
The antibiotic I am using is kanamycin.
I allow the agar to cool down and then add the antibiotic, so I don't think thats the problem.
I was thinking of using a higher concentration of antibiotic.
I did carry out a positive control but I see the same "lawn effect".
Any other ideas?

-researcher-

HI ,
I also face the same problem that u have. Km is not grate antibiotic it tends to degade in 2-3 days. The problem of geeting lawn of competent cells may be due to thehigh cell densities. Try to plate lesses amount of cells on Km plate and see the diffrence. U can also set up a exp where u plate a amount of cell range from 50microliter to 500 microliter on Km plater with concentration which u normaly use. If u want to use higher cell densities u have to use higher Km.
Precautions:
make ur Km plate whenver required ( dont store)
use low densities

Hope this will help U

praveen smile.gif

-praveen_iitb-

In LB bacteria tend to be less susceptible to antiobiotics as compared to e.g. PA.
As such, it is possible that the concentration is too low. At our lab we routinely use 50 mg/l in PA. In addition, for transformation experiments we usually plate out 100┬Ál of the whole transformation mix and in addition plate the rest on another one.
But even w/o dilution a stringent Km selection should yield no bacteria with competent cells alone, and furthermore our plates with AB tend to work even after more than a week storage in the fridge.
Atm the best guess seems to be unsuitable Km concentrations.

-Charon-

thank for all the information
I will try a higher Km concentration and hopefully that will help.
will mail again if there are any further problems
keeping my fingers crossed.

-researcher-

Just a thought, and I am sure you have checked, but is your bacterial strain resistant to the level of kanamycin you are using? Did you check the MIC? Is there any likelihood of spontaneous resistance occurring?

I have had problems myself with transposon mutagenesis and Pseudomonas. It may be the LB agar - if you try Luria Agar as opposed to Luria-Bertani it may work.

I got my TM to work earlier this year but could not replicate, and the only thing I tracked it down to is possibly the difference in agar (thanks to our stores dept switching it on me!). I am waiting with baited breath for some Luria agar to arrive....

Good luck anyway

-Boo-

ok folks
i seem to have eliminated the lawn problem.
i do not see lawns anymore, but on the 25ug/ml Lb-Kanamycin plates with no DNA and only competent cells, I still see a bunch of individual colonies.
I do not think the cells are the problem because:
1) I bought these cells -they are ready to use competent cells (Dh -5 alpha) from Invitrogen
2) I used LB-ampicillin plates @ 100 ug/ml concentration and plated out 100 ul of competent cells only ( i put these cells through the whole transformation protocol)
and I see no growth.
So the competent cells are fine.
Why is it that I see colonies in the LB-Km plate and not in the LB-Ampicillin plates in the negative control?
its strange.
I did borrow a plate of LB-Km 25 ug/ml from some one in my lab and plated out 100ul of competent cells alone and i see no colonies, so obviously I am doing something wrong with the plates.
I use an LB-medium broth 25g/liter of distilled water and 15g of agar, autoclave that for 15-20 minutes as recommended.
I then allow it to cool down considerably ~ 45 mins to 1hr when I am able to touch the flask with my hand and then I add antibiotic @ a final concentration of 25ug/ml from a 10mg/ml stock solution which is freshly prepared and sterile filtered. (So I add 2500 ml or 2.5 ml for 1000 ml of medium..that is right?)
I allow that to mix very well for 10 mins and then pour the plates with the flame on.
I do not know what I could be doing wrong.
I am really close to making this clone work and I hate that this technical problem is delaying everything.
Any help will be greatly appreciated.
Thanks in advance.

-researcher-

One more thing.
I searched online and it says that the powder needs to be at -20 degree C.
does that make a difference? I mean is it possible that it has undergone degradation which is why it is not as effective?
My lab unfortunately stores it at RT.
Please Help...any advice?

-researcher-

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