Hi

What protocol for plasmid-DNA extraction are you using? 

Hi there,

If no plasmid can be extracted from the clones selected by the antibiotics it means your recipient strain is either naturally resistant to this antibiotics or your strain is infected with resistant cells. Do you plate recipient strain alone on selective medium as a control?

The recipient strain can not be resistant since you will get millions of "colonies" and there is no way you can pick them. I agree it is possible that resistant cells are present while it is unlikely for most of commercially available competent cells. You may try a negative control to test the cells if you do not want to switch to another pool of competent cells.

One possibility is that your antibiotic is not properly mixed into your agar (so the antibiotic is not homogenous over the plate allowing growth of some non-resistant cells).

This may be unlikely as rather than discrete colonies you would get bacterial patches.

Some antibiotics are temperature sensitive and break down > 65oC too.

A good tip is: do not add the antibiotic to the agar if the bottle is too hot to pick up comfortably (human's can stand temps < 65oC). Also add the antibiotic slowly with gentle swirling (the ab solution is generally at room temp so if a large amount is added to quickly it can "set" the agar in clumps which can sequester some of the added antibiotic).

Also alongside your transformations add a negative (no plasmid) and positive controls (cells transfected with uncut vector plasmid). The negative control will indicate if there is a problem with your competent cells/agar plates  (there should be no colonies on it) and the positive control will indicate the competence of your host bacteria.

I am not familiar with the plasmid pMRS127. Does it contain the Lac Z gene?

If so Blue-white selection may be the way to go.....

sorry I cant be more help,

Gary

Thanks very much for your responses. I am using a Qiagen mini prep kit for plasmid isolation which normally works very well for extracting plasmid DNA. I shall try the transformation again with the positive and negative controls and also remake the antibiotic plates to make sure the selection is working.    

You might used old plates where antibiotic may be degraded. Try to use new plates. Moreover when you pick up colonies, try to avoid satellite colonies (very small colonies), but choose the well-defined ones. Did you incubate your bacterial culture over 12-16 hours?

If the antibiotic is heat sensitive one can add the antibiotic solution by pipetting onto the plate, and using a plate spreader (glass, L bent) that has been sterilized by placing end of spreader into 100% etoh and lighting with a bunsen burner, then allowing 30 seconds to cool.  I haven't done this with top agar added but have done with plates that I forgot to add antibiotic.  You can try it on a sample plate with a control first so as not to waste your transformants.  I would be gentle on the spreader.  Make sure that the solution volume is adequate to spread on the plate evenly.

This sounds rather as if you are selecting low-level antibiotic resistance mutants, rather than transformants. These arise quite readily if the incubation period is long (>3 days) or the antibiotic concentration is low (

Thanks for all your suggestions. I have prepared fresh plates with the antibiotics taking care to cool down the media sufficiently before adding the antibiotic (200ug/ml Erythromycin) and mixing really well. There was no growth in the negative controls. Also the number of colonies I got was less in the diluted ligation mixture.

 On carrying out plasmid extraction of these colonies, I got a good DNA yield and they also tested positive for PCR of the insert region. But unfortunately, I cannot observe a definite plasmid band when I run the DNA that was extracted. All I see is a smear. Can somebody suggest a solution?

Are you pooling colonies for the plasmid extraction?  I have always picked individual colonies, streaked each colony with a toothpick, and grew each isolate separately to keep the culture pure.  I grow up individual cultures and test them separately (or pool them, for example when I was screening cosmid inserts for a particular set of antibiotic genes and I had a probe that I could used on the various pools.   I only pool purified cultures to narrow the field. I guess because I am trained as a microbiologist.  It may seem like a lot of trouble to do it that way (unless you already are), but I can't understand why you would get a smear if you are (doing it that way), and if you do them as individuals you should get exactly what you want.  I can only think that the plasmid is unstable in your host bacteria, your plasmid prep is contaminated with DNAse, or there are an infinite number of plasmids of approximately the same size in your host strain causing smearing in the plasmid prep.  I haven't worked since 1989, so I am sure that there are other explanations, but if I knew step by step how you were doing your experiment instead of making assumptions on how I thought you were doing it I might be better able to identify the problem.  I am sorry that I cannot help more.

Thanks very much for your answer, any help is welcome. Yes, I am picking individual colonies and culturing them overnight for plasmid extraction. But it maybe that the plasmid is unstable in the host after transformation like you suggest or there is no plasmid and I am just getting genomic DNA. I have been extracting the plasmid of another strain at the same time and following the same procedure but the other one works perfectly.    

Your strain sounds suspect.  If you can find another strain that would fulfill the required characteristics that you desire, but wouldn't chew up your DNA, I would encourage it.