nested PCR

[m397n2002]

Hi Everybody,
Has any of you ever read in papers or tried to run two same PCRs (same primers, same mixture,
same thermal cycling) as a first and second round instead of nested PCR (amplifying a long fragment
in first round and smaller one in the second round)? I mean that do you think it is
necessary to start amplification with the longer fragment first?
thanks,
Mary

[r.rosati]

Yes I tried it when I was young and inexperienced, and yes you really need to change primers. It was a mess, because every slight unspecific product gets amplified heaps in the second round.

[relaxin]

Yes, you need to use primers in the first round to give a longer fragment. Then in the second round, you need to use primers based on the sequence of the first amplicon to give you a samller fragment. Using the same primers as the first round in the second PCR will not give you a smaller fragment.

[procopio f.a.]

the way you were talking about works super well in case of realtime pcr. I mean you run your first pcr then the second one with a taqman probe that will give you the specificity of your product.
If your pcr are instead preparative you should use the classical nested approach, external and internal primers.
fra

[relaxin]

the way you were talking about works super well in case of realtime pcr. I mean you run your first pcr then the second one with a taqman probe that will give you the specificity of your product.

Why not just run one PCR with the TaqMan probe for 40 cycles?

[mchlbrmn]

I agree. With 40 cycles and good efficiency a single copy should be (theoretically) detected.
If you do a second run with the same primer set, dilutions to show the primer efficiency should definitely be run, I would think. Otherwise you will only detect the desired product with the probe, but secondary nonsense products could easily be present but undetected at such extreme cycle number, and the efficiency could be impaired and the quantitation thrown off.