TOPO TA CLONING KIT - (Sep/17/2006 )
i have just started with cloning for the first time and hope that you can give me some useful ideas on how to work with it.
here are some of the questions i need help on. Thanks in advance!
1) the kit says that i have to use *fresh pcr products*. i wonder if i can use frozen pcr products as well? Will the TOPO TA cloning still be effective as with the fresh ones? if i can only use fresh pcr products with this kit. How can i do to save my pcr products? Right now, i only use once and then discard the rest. this will cost me alot. 
2) after i have mixed TOPO TA vectors and inserts. How long can i store them at -20C?
3) how long can i keep the transformation mixture ( transformed E.coli bacterias with recombinant vectors) in the refrigerator (about 4C degrees)? will the mixture be effective as new made?
4) how long can i store transformants in agar plates in the refrigerator (about 4C degrees) before they eventually lose their vectors?
About PCR:
5) i plan doing a PCR directly from bacteria colonies. How long can i store (at -20C) the bacteria in water for PCR?
6) After i have added the bacterias in water and put them at 99C for some minutes and then put directly on ice. After this step can i store them at -20C for later work or do i have to proceed working?
Hi mioarray!
Although I can't answer all of your questions exactly I can say some things about them.
1) Although fresh PCRs are recommended you can use older ones. I already used a some week old PCR without having problems...
2) How long exactly you can store it I don't know - but you can after all. 
3) You also can store your transformation mixture but there will be loss of effectivity. Already after one day storage I made the experience that I had to plate a much higher amount of mixture to get colonies.
4) Here I think you shouldn't wait too long - fungi sometimes grow earlier than you expect them to grow. 
About loss of vector... hm, I once tried a one month old plate again and it was okay.
5)/6) About the storage of the bacteria in water before PCR etc. I might only guess that the lysated bacteria still can be used for PCR after storage. But if I am wrong someone else will let me/us know I guess...
Greetings,
Chakchel
2) one could store the ligation mixture in -20C for a very long time. Sometimes I would leave the ligation on the bench and would transform after a week and it would still b fine. But better store in -20C.
thanks a bunch! 
I have come this far and these are the things i am troubled over. Please, give me your ideas. Thanks alot!
1) when i add the inserts into the vector. the kit says that i have to mix gently. How do i do it? Should i pump the mixture up and down gently or is that enough that i knock the tube gently with my hands and then swirl it around? What problems can i have if i do not mix it gently?
2) when i add the recombinants vectors into the cells, i also have to mix it gently. Any resons why it should be this way?
3) I have got some white colonies. However, i can see that there are more small white colonies than useable size. Why are these colonies that small? 
How can i do to increase their size so i can use them for analyzing? I put the plates back to 37C overnight, but it does not help. They still are at this little size, so they are useless for working. 
Hi!
Why to mix the ligation mix gently I can't tell you, I never had problems mixing it the "usual way".
The cells have to be treated carefully because they are quite sensitive. Their membrane is very porous and mixing by pumping up and down could make them break... That's is also the reason why you have to thaw them slowly on ice.
Just add the ligation mix to the cells and stir carefully with the pipette tip. Then knock the tube carefully with your fingers. That might be enough to mix everything properly.
Do you get some "big" white colonies, too?
Maybe the small ones that don't grow anymore are satellite colonies?
Greetings,
Chakchel
Why to mix the ligation mix gently I can't tell you, I never had problems mixing it the "usual way".
The cells have to be treated carefully because they are quite sensitive. Their membrane is very porous and mixing by pumping up and down could make them break... That's is also the reason why you have to thaw them slowly on ice.
Just add the ligation mix to the cells and stir carefully with the pipette tip. Then knock the tube carefully with your fingers. That might be enough to mix everything properly.
Do you get some "big" white colonies, too?
Maybe the small ones that don't grow anymore are satellite colonies?
Greetings,
Chakchel
Thanks again!
This is a silly question, but how do you thaw the cells slowly on ice?
There are no silly questions...
Well, I just take the cells out of -80°C and put the tube immediately onto ice, there I let them thaw for 10 - 20 minutes. Then they are ready to use.
(Slowly in this case means that you shouldn't thaw them in your hands or at room temperature)
Chakchel
the cells are mixed in with glycerol which lowers the freezing point of the cell suspension.
Well, I just take the cells out of -80°C and put the tube immediately onto ice, there I let them thaw for 10 - 20 minutes. Then they are ready to use.
(Slowly in this case means that you shouldn't thaw them in your hands or at room temperature)
Chakchel
Thanks alot!
