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TOPO TA CLONING KIT [View Printable]
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user
Group: Member
Posts: 29
Joined: Jul 07, 2005
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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?
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| Posted Sep 17, 2006, 14:08 PM |
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vasussci
Group: Member
Posts: 37
Joined: Aug 14, 2005
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| 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.
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Ian Taylor
find.compare.save.
www.vasus.com
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| Posted Sep 21, 2006, 20:49 PM |
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montgomj
Group: Member
Posts: 97
Joined: Feb 22, 2005
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| 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.
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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 |
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| Posted Jul 03, 2007, 18:03 PM |
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parvoman
Group: Member
Posts: 256
Joined: Jul 28, 2005
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| 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.
XXX The kit I use involves only a 5 minute ligation so it's easy to do it straight after the PCR. If you're worried about loosing the overhang then use a Blunt cloning kit (together with a proof reading polymerase for the PCR)XXX
2) after i have mixed TOPO TA vectors and inserts. How long can i store them at -20C?
XXX -20 for a few weeks (but you shouldn't need to because the ligation reaction is very efficient XXX
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?
XXX I usually keep about 90% of the transformation mixture (bugs/LB) in the fridge overnight until I see that the 10% plated out yielded colonies. I never need it though but hate throwing stuff away to early. If you have reason to believe that there will be very few colonies then I would plate out ALL of the transformation mixture on up to 10 LB amp plates, which you could also pre-warm to 37 just before plating out. XXX
4) how long can i store transformants in agar plates in the refrigerator (about 4C degrees) before they eventually lose their vectors?
XXX If the colonies are well spaced out and the plates were poured with quite think agar (20mL) then they should last quite a long time. I use Parafilm to seal the petri and store at 4 degrees. If the colonies haven't dried up then you should be able to re-grow the bugs/plasmid in LBamp for around 2 months (although I would not choose to wait this long - better to do a glycerol stock).XXX
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| Posted Jul 20, 2007, 20:54 PM |
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elayabalan
Group: Member
Posts: 1
Joined: Nov 03, 2007
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i am aslo doing TOPO cloning with same proplem,i got only few colony but no amplification in colony PCR,what i can do, kindly suggest me |
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| Posted Nov 04, 2007, 17:58 PM |
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parvoman
Group: Member
Posts: 256
Joined: Jul 28, 2005
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The TOPO kit is usually very efficient. If you only have a few colonies then check that the PCR polymerase you are using is the correct one for the kit. ie. TOPO-TA kits should be used with non-proofreading polymerases such as taq and TOPO-blunt kits with proofreading enzymes such as Pfu or Vent etc. If you can see a PCR product on the gel then you should be able to get a TOPO vector to accept it.
If the kit is quite old and has been thawed and frozen too often then it could be that the topo on the ends of the vector have degraded, preventing efficient ligation, but I've used kits that are 2 years past the use by date and they still worked (albeit less efficiently).
I had a chat with a Clontech rep today and came away thinking that I should maybe try using their "In-Fusion" product which allows you to clone PCR products directly into your vector of interest, rather than via the TOPO intermediate constructs.
http://bioinfo.clontech.com/infusion/
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| Posted Nov 07, 2007, 21:09 PM |
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