It is described in the attachment...
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#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...
It is described in the attachment...
#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!
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!
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.
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.
