control DNA transformation- using pGEM kit - transformation (Feb/02/2011 )
I am very new to this forum and cloning. I am working on a school project on cloning. I am only seeing white colonies on the control DNA plate, it means that my control worked because the control DNA is designed to produce white colonies. I also see white colonies with very faint blue centers on my plate with the inserted ligation. Can I assume that these colonies have my insert. Pls advise .
grow in broth and extract plasmid to check?
uglearner on Wed Feb 2 20:16:23 2011 said:
I am very new to this forum and cloning. I am working on a school project on cloning. I am only seeing white colonies on the control DNA plate, it means that my control worked because the control DNA is designed to produce white colonies. I also see white colonies with very faint blue centers on my plate with the inserted ligation. Can I assume that these colonies have my insert. Pls advise .
Huh.. we never got to do this sort of stuff at school!
I don't know for sure, but I suspect, that the reason for the white colonies with blue only at the centre is because the X-Gal or IPTG got used up and metabolised by the centre of the colony as it grew towards the size which you observed it at. Thus, I'd reason that those colonies do not contain the insert. Still, I've been wrong many times before! As the Curtis suggests, you could find out for sure by extracting your plasmid and restricting or performing junction PCR. Not sure whether your school would provide the resources for this, though?
seanspotatobusiness on Fri Feb 4 18:12:56 2011 said:
uglearner on Wed Feb 2 20:16:23 2011 said:
I am very new to this forum and cloning. I am working on a school project on cloning. I am only seeing white colonies on the control DNA plate, it means that my control worked because the control DNA is designed to produce white colonies. I also see white colonies with very faint blue centers on my plate with the inserted ligation. Can I assume that these colonies have my insert. Pls advise .
Huh.. we never got to do this sort of stuff at school!
I don't know for sure, but I suspect, that the reason for the white colonies with blue only at the centre is because the X-Gal or IPTG got used up and metabolised by the centre of the colony as it grew towards the size which you observed it at. Thus, I'd reason that those colonies do not contain the insert. Still, I've been wrong many times before! As the Curtis suggests, you could find out for sure by extracting your plasmid and restricting or performing junction PCR. Not sure whether your school would provide the resources for this, though?
This was another of those times! I've just read in our Molecular Cloning book (Sambrook and Russel), page 1.125, "Colonies that carry recombinant plasmids do not contain active beta-galactosidase. These colonies are creamy-white or eggshell blue, sometimes with a faint blue spot in the center". So it sounds as though your colonies might very well (have) contain(ed) the desired plasmids!
Still, this raises the question for me, what's going on at the centre of those colonies to produce the blue colouration?
Possible of overlapping between inserted and non-inserted colonies. There is also a vary rare chances that your inserted fragment forming a recombinant proteins which is a weak but active beta-galactosidase gene.
I would do a streak plate to double verify the purity again before using it in my work.