PCR master mix - (Nov/25/2005 )

I bought ready to use pcr master mix solution (2x 1.25ml ) from fermentas. But I am having problem in how to apply it to my own project. The composition of the 2x master mix (fermentas)as below:
0.05u/microliter Taq DNA
reaction buffer
4mM MgCl2
0.4mM of each dntp

But what I used to use in my PCR is :

1.5 u TAq DNA
reaction buffer
2mM of MgCl2
2.5 mM of mix ddntp

I am blur at the moment and I need a lot of help regarding this, I am doing RAPD-PCR on genomic of tilapia (Oroechromis niloticus)

endran

-endran-

What volume are your PCR reactions?

daniel

software for sequencing DNA

-Daniel Tillett-

Used as a 2x master mix the final concs will be (for a 50ul reaction):

200uM dNTP (normal working conc)
2mM MgCl
1.25U Taq (25ul of 2X mm multiplied by 0.05)

You say you use 2.5mM each dNTP when making your PCRs normally, this is the stock conc which will translate to 10mM dNTP mix which is final conc of 200uM

10mM x 1ul / 50ul = 0.2mM

The master mix is fine, go ahead and use it

-John Buckels-

I am using 25ul volume of PCR

-endran-

QUOTE (John Buckels @ Nov 26 2005, 12:47 AM)

Hi,

, I really dont understand.. I am new to this thing and can you elaborate to me again! thank you very much
endran

-endran-

QUOTE (endran @ Nov 25 2005, 02:23 AM)

Whats the problem?
When you have a 2x mastermix, deside the volume, calculate half volume, add template, add primers, add water to half volume (calculated earlier), then equal amount master mix.
or, was something else the problem - specify, please.

-Gerd-

The point of a 2x mastermix is that you don't need to worry about working out individual components - the R&D guys at fermentas have spent months or years developing this product to make things easier for you

For a 25ul PCR use the following

12.5ul of 2x mastermix (fermentas)
1ul of each Primer
5ul of template DNA
5.5ul water

-John Buckels-