Molecular biology master mix preparation question?

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Molecular biology master mix preparation question?

Post by cryocell » Fri Apr 29, 2011 7:59 am

okay guys would I prepare this master mix...

I need 48 μL master mix total which will be added to 2 μL of template to create a 50μL total reaction volume...

how much ...
5x taq buffer would i need _at_ a 1x concentration
taq DNA pol 5U/μL at a concentration of 1.25 U would I need?
Forward Primer 10 μM at 0.2 μM concentration?
Reverse Primer 10 μM at 0.2μM concentration?
and how much nuclease free water would be added?

also 1 μL of nucleotide 10 mM mix is added at a concentration of 0.2 mM to the master mix.

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Post by JackBean » Fri Apr 29, 2011 8:24 am

is that really so hard to calculate? :roll:

If you're pipetting it one solution at a time, than it's no master mix. Master mix is done for example in amount of 1 ml and then aliqouted.

The final volume is 50 ul and you need to
5x dilute buffer
5/1.25-times dilute Pol
10/0.2-times dilute both primers
10/0.2-times dilute the nucleotide mix (which is 1 ul, so that should tell you something about the primers;)
and add water up to 48 ul

Cis or trans? That's what matters.

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Post by stephroche » Fri Apr 29, 2011 2:01 pm

Buffer 10µL
Primers : 1µL each
dNTP : 1µL
Taq Pol : 0.25µL
H2O : 34.75µL

+ 2µL DNA

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