TOPO TA CLONING KIT - (Sep/17/2006 )
I have got an advice on how to "re-use" frozen PCR samples. I am adviced to incubate them with Taq polymerase + buffer+ dNTP for 15 min at 72 degrees. This will re-add A overhangs, in case they are lost. I wonder if anyone knows how to incubate frozen PCR samples with Taq polymerase + buffer+ dNTP? How much PCR products, Taq polymerase, buffer and dNTP i need? 
Has anyone tried this before? Does it help?
Thanks alot!
Make up a PCR reaction just as you normally would, except omit the primers and use more template DNA. Heat to the extension temperature (typically 68 or 72) for 10-30 minutes. If you have dATP (Note: different from ATP!) sitting around, then you can use it instead of the dNTP mix with perhaps marginally better results.
Since it sounds as if you are just starting out, I'll add some unasked for advice -- In general I find it inefficient to attempt to "rescue" failed experiments. If I were doing this, I'd just redo the PCR and move forward. There are more than enough variables to try to control as it is. Adding more from uncertainty about what happened a week ago to that sample I stuffed in the freezer is not one I want to layer on top. I would never store a PCR product in unpurified form for long periods. I would never store a ligation reaction hoping to transform cells another day. You want to start with known-good reagents and samples, work forward, purify, and consolidate your gains (or figure out what went wrong). Good places to stop are with purified plasmid DNA (preferably sequenced!) and with transformed cells containing those plasmids stored as glycerols at -80C.
Since it sounds as if you are just starting out, I'll add some unasked for advice -- In general I find it inefficient to attempt to "rescue" failed experiments. If I were doing this, I'd just redo the PCR and move forward. There are more than enough variables to try to control as it is. Adding more from uncertainty about what happened a week ago to that sample I stuffed in the freezer is not one I want to layer on top. I would never store a PCR product in unpurified form for long periods. I would never store a ligation reaction hoping to transform cells another day. You want to start with known-good reagents and samples, work forward, purify, and consolidate your gains (or figure out what went wrong). Good places to stop are with purified plasmid DNA (preferably sequenced!) and with transformed cells containing those plasmids stored as glycerols at -80C.
Thanks a million, Phage!
hi guys,
i will do cloning for the first time and my prof is really counting on me bout this tuff and this site has helped me clear a lot of things in my mind. however, i cant confirm if it is possible that i can clone my bacteria- sulfur-oxidizing-denitriying (autotrophic and very hard to grow) bacteria with this kind of kit?
and where can i find a detailed explanation of this kit?
thank you very much for the expected replies...
chickoy22