Hi All,
My current work needs to insert a large piece DNA into a vector , it's about 5k bp long. We were used invitrogen HF-tag for PCR many years, and it works very good for 1-2k bp insert PCR, however, it seems very hard to get PCR products over 3k. As this time we need to PCR a 5k product, did anyone has any recommendation for it? I.e different Tag enzyme or condition?
Thanks
Yiyun
You could try another high fidelity polymerase (eg. Phusion polymerase from New England Biolabs). The upshot of Phusion is it runs a lot faster than Taq and is still high fidelity. With it you should have no issues producing a 5kb insert. If you want to stick with the Taq, you could always try an series of overlap extension PCRs on smaller sections of the larger product, or try cloning in your desired insert in steps (1st half, then 2nd half), provided you can find a unique cut site in your insert.
Hi there,
I do agree with the answer above : Phusion polymerase is fast and efficient and adapted to long PCR (up to 20kb fragment amplification).
Dear Monica
I agree that the key is to use a highly processive polymerase exemplified by Phusion, not standard low processive Taq
However, in concert with this enzyme there are things you can do to improve yield of your product by increasing PCR efficiency over the 5 kb
1. Secondary structure by virtue of size is inevitable: Thus add 2-5% DMSO to your PCR or better still 0.5M betaine
2. Start your PCR with an initial denaturation/hot start step of 95C for 5 min: This will activate your enzyme but more importantly will render much of your template single stranded which will greatly improve initial priming, subsequent amplification and therefore ultimate efficiency of your PCR
3. During your annealing keep conditions relaxed as detailed in the suggested program below taken from the attached Qiagen doc on 'long range' PCR; That is TM -5C which will encourage highly efficient priming
Base the Tm of your primer and thus your annealing temp (Tm-5C) on a program which incorporates your actual PCR conditions into your Tm calculation and not just the standard nearest neighbour algorithm which simply takes account of base pair composition and skewing, e.g.
http://www.idtdna.com/calc/analyzer
4. Another factor to consider is to ensure that you do not have rate limiting quantities of either polymerase or dNTPs in particular. The latter will limit PCR efficiency if too low by simple mass action. Thus as detailed below make sure your [dNTP] = 500uM, i.e. ~ 2 x standard concentration and similarly use 2 x the standard amount of polymerase
In addition, consider using a modified buffer with additives that also help stabilise your polymerase and increase its half life
5. When denaturing keep your temp @ 95C rather than the std 96-98C (recommended for example with Phusion): This will limit denaturation of your enzyme in conjunction with DMSO plus very high denaturation temp (>96C) which can affect even hardy polymerases like phusion and also de purination of your parent DNA template strands
6. By extending for prolonged periods, e.g 5 min per cycle @ 72C, DNA template stands can begin to de aminate, which poisons your PCR and limits efficiency: Thus extend @ 68C instead
Thus:
Initial activation step: 3-5 min 93°C Initial denaturation of template DNA.
3-step cycling:
Denaturation 15 s 95°C Do not exceed this temperature.
Annealing 30 s 62°C Approximately 5°C below Tm of primers (see Appendix B, Table 13, page 29).
Extension 1 min/kb 68°C Use an extension time of 1 min per kilobase DNA for genomic DNA targets. There fore use 5min @ 68C
Number of cycles 35 Amplification for 35 cycles will produce satisfactory results in most cases. However, the optimum number of cycles depends on the amount oftemplate DNA. See Appendix C page 32 for guidelines.
72C 15min x 1
End of PCR cycling: Indefinite 4°C
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