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PCR Reaction


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3 replies to this topic

#1 LanMolecular

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Posted 13 February 2012 - 11:10 PM

Hi everyone. Anybody knows if my method is suitable for amplification of bacterial plasmid DNA?
It is described in the attachment...Posted Image

pcr for u.png

#2 Julio-Claudian

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Posted 14 February 2012 - 03:40 AM

Hi LanMolecular,

Is this your first amplification or a "recipe" passed down to you?

Reason I asked is that the final concentration of Mg2+ (3mM) is quite high for me. For Taq, I usually add it into the individual tubes before starting the cycles. I add the primers into the master mix though.

I have no complains about the cycles and temperature but depending on the outcome, the annealing time [and temperature] might change.

Unless this is a repeat of what was done before, you might need some tweaking after this.

Happy working! Posted Image

Edited by Julio-Claudian, 14 February 2012 - 03:58 AM.


#3 LanMolecular

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Posted 14 February 2012 - 08:41 PM

Well, it is a secret recipie that is passed down to me. I'm new in technically but not in theory... So, what is the possible amount of Mg2+? 4 microlit? I'll used the master mix for 4 pcr tube... And thanks for reply.

#4 Julio-Claudian

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Posted 14 February 2012 - 09:15 PM

It's good that you're familiar with the theory behind the workings of each components.

Since it's a passed down secret recipe, I'm sure it's been optimised (?). So I think there must be a reason for such values. In any case, perform the amplification, see what you get, and work from there.

I remember starting my Mg2+ at 1.75 mM and [together with other factors] went up until 2.5 mM. But like I said, it depends.




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