yeast contamination - problem in cloning (Sep/20/2005 )
I am facing terrible problem in cloning. The DNA restriction looks proper and even the ligation reaction when run on gel shows gel shift as compared to vector backbone alone. During transformation I get only a few colonies and most of them donot grow in culture. Examination under microscope shows circular bodies (I believe them to be yeast). SHOULD IT BE ADVISABLE TO USE NYSTATIN IN LB PLATES TO REDUCE THE YEASE BACK GROUND. One person in my lab is working with yeast.
please reply I an utterly disappointed.
hi
i'm not sure of yeast contamination... i suggest you few things by importance order...
Test first the efficiency of tranformation of your colonies with a well established vector from your lab and pour your own new plates. you'll gonna see efficiency and be sure of the antibiotic concentration.
Second decrease the amount of selective antibiotic you use on your plate.
Third, try fresh made competent bacterias
Fourth change bacteria strain...
fred
I am not sure about your conclusion. If you are using standard sterile microbiology procedures, then you shoulod not be having any problems with yeast contamination. I recommendend using a positive control test plasmid to see if your transformation procedure is not working.
What is your selection antibiotic? Are you allowing a non-selective, post-transformation incubation period to overcome phenotypic lag if it is required (which depends on the antibiotic you're using)?
I agree with the suggestions above -- test your cells to be sure they're competent (is this a transformation with chemically competent cells?), and pour new plates.
Since you have gel evidence of a successful ligation, and are not getting transformants, less-than-optimal competent cells would be my first guess, followed by some selection issue (like using a too high antibiotic concentration). Only after we've ruled these easy things out do you start to think of other, more troublesome things (like lethal genes, plasmid incompatibilities, etc.).
I agree with the suggestions above -- test your cells to be sure they're competent (is this a transformation with chemically competent cells?), and pour new plates.
Since you have gel evidence of a successful ligation, and are not getting transformants, less-than-optimal competent cells would be my first guess, followed by some selection issue (like using a too high antibiotic concentration). Only after we've ruled these easy things out do you start to think of other, more troublesome things (like lethal genes, plasmid incompatibilities, etc.).
The cells are competent as I get countless colonies by plasmid transformation. the antibiotic is kanamycin whose concentration on plate is 40µgm/ml. I have changed plates many times. plasmid incompatibility should not be a problem as this is the only plasmid in cells. the genes are under t7 promoter so I guess the translation product of gene (which might be toxic ) is ruled out.