Hot start polymerases and normal taq - (Dec/17/2006 )

Hi there,

I have been using a hot start polymerase for my PCRs, requiring an initial 7 minute activation step.

Its quite expensive, and I will be using it a lot, so I would like to trial other standard taqs alongside it.

My question is, if I use a PCR program with an initial 7 min at 95 degrees, will it damage or otherwise interfere with the activity of a normal taq polymerase?

thanks

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Should be okay I think.

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absolutely fine, have you conducted colony PCR ?? I even use 10min 95 in the first step,

but what is the use of 7 min 95 when you just using normal Taq ??? it is meaningless

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an addition to lactamase comment, since you are no longer using hot start, you can drop the initial 7min melting step. Of course if you are using this for colony PCR, you do neet an intial heating step to lyse the cells. I use 5min in this situation, rather then 10 min which Lactamase uses.

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Oh, I forget to tell somthing more detail, for B subtilis, I use 10 min as I find that 5 min sometime don't work well (is that due to the relatively thick cell wall of Gram positive bacteria?)

I also use 5 min 95 oC in the case of E coli

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probably....

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Thanks a lot for that,

I will try it out. I would like to run both polymerases on the same assay plate initially to compare results. If the normal taq is just as good for my purposes I'll drop the initial step.

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extent preheat step will reduce efficiency of normal Taq
you could use " hot start" PCR program for normal Taq, such as preheat PCR to 90C before add PCR tube.
the reason using hot start, most time, is for reducing non-specific amplfiication, I think

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