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?

vasussci said:

user said: 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. They guarentee the integrity of their kit and the likely hood you will have successful ligations with fresh product. Also old products will lose the needed A overhangs needed to ligate with a TA kit 2) after i have mixed TOPO TA vectors and inserts. How long can i store them at -20C? should be fine for a while, but I would do this for a more than a couple weeks. You product will degrade if there is any contamination and the efficiency of transformation will decrease. 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? few days. You can have loss of plasmid and cell death quite easily. If inoculating a fresh culture make sure you add you seletive antibiotic. If you want a quick frozen stock, add glycerol to 10-15% and store at -80. 4) how long can i store transformants in agar plates in the refrigerator (about 4C degrees) before they eventually lose their vectors? Transformants will last longer as single colonies on a plate than in liquid, but after a week or so you're antibiotic will break down and loss of plasmid will occur.

RE: 1) - You can thaw frozen PCR product, incubate it with Taq and dATP for 15 min at 72 C to add the A overhangs back and then use it in your TA ligation reaction