Transformation troubleshoot - (Jul/22/2008 )
Hi,
I'm trying to isolate a plasmid from yeast and then transform it into bacteria.
I used our lab's protocol to isolate DNA from yeast. At the end of the protocol, I checked the OD and then proceeded to use 5ul in a tube of 100 ul competent DH5alpha cells. Out of 10 different transformations, I might have gotten one colony on one plate.
My question is: I added 5ul of DNA from the yeast prep regardless of the concentration of the DNA. The concentrations ranged from 640 to 2200ng/ul. Am I using too much DNA in the transformation? I know with bacteria miniprepped dna, we use a lot less DNA.
Also, if I didn't see any colonies after an overnight incubation, is it advisable to leave the plates in the incubator for another day to see if there is any growth?
Thanks!!
Paige
I'm trying to isolate a plasmid from yeast and then transform it into bacteria.
I used our lab's protocol to isolate DNA from yeast. At the end of the protocol, I checked the OD and then proceeded to use 5ul in a tube of 100 ul competent DH5alpha cells. Out of 10 different transformations, I might have gotten one colony on one plate.
My question is: I added 5ul of DNA from the yeast prep regardless of the concentration of the DNA. The concentrations ranged from 640 to 2200ng/ul. Am I using too much DNA in the transformation? I know with bacteria miniprepped dna, we use a lot less DNA.
Also, if I didn't see any colonies after an overnight incubation, is it advisable to leave the plates in the incubator for another day to see if there is any growth?
Thanks!!
Paige
Not sure what kind of protocol you're using to prep your yeast DNA, but when I used to do this the quality of the DNA was always a lot lower, so I didn't worry too much about adding extra because I didn't trust the OD readings. If you're concerned about it, you can always do a couple of tranformations at once, reducing the concentration 2x for each one. I would not let the plates grow for another day. Your bacteria should grow just fine within a single day and all you will do is add contamination to your plates.
How much would be too much though? Do you know if there's an optimal range? Could I check the quality of the yeast dna prep on a gel first? Sorry for all of the questions but the boss is out of town. :-)
How much would be too much though? Do you know if there's an optimal range? Could I check the quality of the yeast dna prep on a gel first? Sorry for all of the questions but the boss is out of town. :-)
You could certainly try running some of the DNA on the gel if you wanted to. I would suspect that you will see a lot of large molecular weight material that would correspond to the genomic DNA, again it would depend on what kind of protocol you were using to isolate the DNA in the first place, but it might give you some indication of how pure it is. I think for a normal plasmid transformation you use around 200 ng of DNA. I looked back at my yeast DNA isolation protocol and all it says is to try transforming between 1-5 ul of DNA. I recall that it was often tricky to get the plasmid back into bacteria - I would get very few transformants. I do believe that some companies sell kits that make it easier to extract plasmid DNA from yeast, but I haven't tried them. I remember that one member of this forum was doing a yeast two hybrid screen recently and he was doing transformations with it. Perhaps he can give you more information.
Here is a link to that thread.
http://www.protocol-online.org/forums/inde...hybrid&st=0
Good luck to you.
smu
How are your competent DH5alpha cells prepared? What is the size of the plasmid you're trying to transform them with? What is the selectable marker on your plasmid? What is your transformation protocol? Do you allow for phenotypic lag if applicable? If you run a "competency control" by, say, transforming the DH5alpha with pUC18 or some similar E. coli vector, do colonies arise?
competent cells were made in the lab, by a CaCl2 protocol, I believe. The plasmid is from a pGAD with an unknown insert. I'm going to attempt to sequence the insert. The vector itself is 8kb with an amp selection. Transformation:
Competent cells on ice 5 min to thaw
Add 5ul yeast prep dna. Sit on ice 30 min.
Heat shock at 42 degrees for 90 seconds.
Add 1 ml LB. Incubate at 37 degrees for 1 hour.
Spin down, remove majority of supernatant, resuspend pellet and plate on LB+amp plate.
I'm not sure what you mean by phenotypic lag. And I haven't done a competency control. I'll see if I can get a hold of some vector as a transformation control.
Thanks!
Well, an "unknown insert" can mean a lot of things -- I'm mostly concerned with the size of the ultimate plasmid you're trying to use for transformation (transformation gets tougher as the plasmid gets larger).
For preparation of competent E. coli, I use a rubidium chloride method that produces very competent cells -- see here. Yikes -- I wrote that page 13 years ago!).
Definitely do a competency check -- if your cells aren't competent (or are insufficiently competent), you'll never get anywhere...
For preparation of competent E. coli, I use a rubidium chloride method that produces very competent cells -- see here. Yikes -- I wrote that page 13 years ago!).
Definitely do a competency check -- if your cells aren't competent (or are insufficiently competent), you'll never get anywhere...
Thanks for the help!!!!