moe, a CAT gene question for you

[tinker bell]

Moe!
I'm trying to clone a gene that has two different drugs selection. One is Amp and the other is CAT. I cut the CAT gene out of pkD3 and put it into a vector called pUC19. I always get clones on the Amp plate but never get anything from the CAT plate. However, when I streaked pKD3 straight on a CAT plate, it grows just fine. I'm sure that my CAT plates are fine. It's just so hard to manipulate the CAT gene. Have you ever experienced anything like this? Any inside clues would be greatly appreciated. I can't ask any one because no one is a CAT expert here. At least all those people I know around my department.
Thanks a lot

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[moe]

OK, so let me make sure I understand your question. You are cutting the CAT gene out of pKD3 and trying to clone it into pUC19 which already has Amp resistance on it. After transformation, you are only getting colonies on Amp plates and none on Cam plates correct? Let me know if this the problem before I give a complete answer, so I answer the proper questions. Also, what concentration of Cam and Amp are you using just for my own knowledge?

 

[moe]

TinkerBell, one other question. What organism are you trying to put this plasmid into? E. coli I would guess.

 

[tinker bell]

Moe!
Thanks for responding! You got the question correctly.
Amp conc.= 100 microgram/ml
Cat conc.= 20 microgram/ml
I transformed them into E.coli DH5alpha and JM109 and both yield the same result.
In short, I just couldn't cut the CAT gene out to insert it into any other vectors....even with the TOPO cloning kit from invitrogen when I just PCR the CAT gene and put it into the TOPO VECTOR, nothing grew on the cat plate as well
T

 

[moe]

Tinker Bell,
It could be a couple of things going wrong here. First off let me tell you that Cam is a bad selection agent. Even when you have the concentration within the proper range it always seems as though you get a lot of background. Usually if I was in your shoes I would use Amp as the selection agent and then pick colonies to a Cam plate to see if any grow. The ones that grow should then have your Cat gene. Before you do that however, you need to change the concentration of your Cam plates. 20u/ml seems high to me. The highest we ever use in our lab is 10u/ml and sometimes we only use 5u/ml. Since you are having problems, I would probably start out with 5u/ml and then see if the colonies that grow on that can tolerate 10u/ml. Anything that grows on your Cam plates I would do a restriction digest of the plasmid with the same enzymes you used to put gene in the vector, and then run it on an agarose gel to see if you get back the two fragments you want. These would be the vector and the gene. If the sizes match up then you most likely have the gene. You could always do a plasmid prep from a couple of your Amp colonies and do the same digest on them. This would then tell you if they have only the vector present, which you know must be there b/c they are growing on Amp or if they have both thevector and the Cta gene. The thing that sticks out most to me is that you are using 20u/ml Cam plates and this seems very high to me. I think that if you bump down the Cam to 5-10u/ml things will work much better for you. So there you have it, that is my .02. Let me know how things turn out.
moe

 

[tinker bell]

All right!

Thanks for the inside scoop! I can't believe it, the book said the range for cat concentration is 40ug-170ug. Holy conc.

I certainly will go back to this and let you know how it turns out in a few weeks. I'll probably streak out hundred of clones on a small cat plate to see if they could grow.
Great suggestion!
Thanks again
T