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Efficient cloning of PCR products.
(Note that the kit details a slightly different procedure which may give superior efficiencies.)
- Mix 1 μL PCR product (amplified with Platinum PCR supermix) with 0.5 or 1μL TOPO vector (0.5μL should be sufficient.)
- Incubate 5 mins on the bench top
- Place on ice
- Tom Knight claims you can cut the reaction volume in half and it will still work fine.
- Use fresh PCR product for best results. (The A overhang tends to get chewed back over time ... even using day old PCR product can reduce efficiency.)
- Your primers should NOT have 5' phosphates. They interfere with topoisomerase I.
- From Tom Knight: As this article discusses, the 5' end of the PCR primer has a strong effect on the likelihood of 3' A addition to the PCR product when using Taq or Taq mixtures as enzymes during PCR. Since it is often necessary to add such an overhang to PCR products for cutting of an added cloning site, and since for such applications the sequence is immaterial, we should probably standardize that sequence to the A tail favoring sequence, GTTTCT for all PCR primers we make. This will favor a 3' A overhang on the PCR products, and allow TA cloning, or TOPO TA cloning of these products. Blunt end cloning could still be done by using Pfu or Phusion enzymes. I can't make any sense from the result about extending the primer sequence another base which is not specified. It seems to me this can not have any effect.
- The 3' A overhang efficiency can be promoted even further by increasing the sequence length to GTTTCTT.
- Reshma Shetty was generating some PCR fragments via annealing and primer extension and trying to cut with BioBricks enzymes and clone but the reactions were failing. Switching to TOPO TA cloning solved the problem.
- In order for your part to be in BioBricks format after TOPO TA cloning, you must have the full BioBricks ends on your primers.
- Austin Che observed anecdotally that he had a greater percentage of correct clones when he screened colonies from Kan plates rather than Amp plates.
- Also see Knight:Addition of 3' A-overhangs.
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- US Patent 6,916,632; Methods and reagents for molecular cloning. (On topoisomerase-mediated cloning using a 5' overhang and blunt end.) pdf html
- US Patent 5,766,891; Method for molecular cloning and polynucleotide synthesis using vaccinia topoisomerase pdf html
- US Patent 6,653,106; Topoisomerase based ligation and cloning methods pdf
- US Patent 6,548,277; Method for molecular cloning and polynucleotide synthesis using vaccinia topoisomerase pdf html
- US Patent 5,487,993; Direct cloning of PCR amplified nucleic acids pdf html
- US Patent 5,827,657; Direct cloning of PCR amplified nucleic acids pdf html