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Cloning with real time PCR fragments - using SyberGreen from Applied Biosystem (Apr/24/2006 )


my boss wants me to clone my real time products, I got with syber green for sequencing.
My problems are that I don´t know which Polymerase this SyberGreen uses (A-overhang or not) and that my pcr products are quite small (70 - 130 bp). I tend to use a TA cloning kit, but I don´t know what would be a good strategy/protocol. Besides I wondered if nested PCRs wouldn´t do the job faster, but somehow my boss is an old technique enthusiast :/
Any suggestions?
Thanks in advance.



Well, most likely your polymerase will be some mutant of good ol' Taq. Most enzymes not having an overhang are proof-reading enzymes, and I don't see why any company would include a proofreading enzyme in a real-time PCR reaction.


Yes, the Qiagen one, at least, is Taq based and produces A overhangs. I'd be surprised if others are significantly different.


It is easy
I use Qiagene PCR purification kit to purifiy PCR products generated by ABI sybr green mix (even 60bp amplicons can be purified by this manner), you might also try recover your products' band from agrose gel using Qiagene gel pruification, then use TOPO (TA) cloning kit for cloning and subsequent sequencing.