I have synthesized cDNA in my lab using Clontech SMART cDNA synthesis kit. I am trying to insert the cDNA into TA vector. However, this didn't work. Could anyone suggest or comment on the idea of cloning cDNA into TA vector. Would it work?
TA vectors rely on the 3' protuding -A left by most thermostable non-proofreading polymerase, such as TAQ. Does not work with proof reading polymerase, such as pwo, pfu and many others. Does not work after polishing protocols, such as T4 polymerase for flushing ends. In addition, irrespective of the PCR amplification method used, the protuding -A can be lost easily. A good trick is to rescue the protuding A of your purified PCR fragment by a 10-15' treatment at 72 °C with a few units of TAQ (or similar) polymerase + dATP (say 0.2 mM ).
Thank you Maurizio for the answer. The kit uses a Platinum Taq pol which incorporates an A overhang. Would the treatment make the protruding 'A' stable, and if so, how long?
In general, it is suggested to use the PCR product as soon as possible after synthesis, since the overhang can be degraded overtime by the polymerase itself. Storage at 4C should be avoided as much as possible before TA cloning.
The other issue I can think of is the insert size. cDNA are usually quite long, how big is the insert you are trying to clone? TA cloning has a very strong bias for small size, meaning that the longer the fragment, the less efficient the cloning. Especially, if there are other smaller molecules in the mix, they will get cloned preferentially. If you are dealing with a large fragment you might want to consider kits optimized for large inserts. We used the Invitrogen TOPO XL succesfully, although not from cDNA.
Hope that helps.
As Andrea remarked, the sooner the better. You can never know. As a rule of thumb either you use your PCR product immediately or add some more A prior to ligation
Well, to be precise TOPO cloning is still TA cloning, is just more efficient. I was suggesting to use the XL version of TOPO TA cloning, in case the insert is very long.
Didn't you get colonies or you just had few white vs blue colonies? I would also suggest to verify the competence of your cells. With libraries I usually buy TOP TEN (Invitrogen)
Barbara I got some blue and few white colonies. The competency of the cells I use is good. I have used them for earlier experiments and I have got good results.
but if I understnd correctly even the white colonies you had did not have the insert. Most likely what happens is that you stored for a long time the vector, or at -20, or even worse, with a "modern" freezer that goes down to -30 but is opened a lot during the day and has cycles of -30 during the evenings and -20 during day time (which means cycles of freezing-thawing for enzymes, etc) and you have lost the Ts on the vector, thi will give you the white colonies (without insert)
Correct me, if you say cDNA then it is a DNA-RNA hybrid and not a double stranded DNA. The RNA DNA hybrid might not have good end to ligate.
One solution is to make it a double stranded DNA with one or 2 cycles of PCR with degenerative primers.
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