Simple question of dNTP's for PCR - (Dec/12/2011 )
So simple question. I'm at a pretty big lab, so the amount of instruction I get is limited as everyone is really busy. I'm also new, and I don't want to keep pestering the post docs with questions. I asked one of the post docs where the dNTP mixture was for the PCR and she gave me a box, but all it had was individual 100mM dATP, dTTP, dCTP, and dGTP from invitrogen:http://products.invitrogen.com/ivgn/product/10216018
I need 10 mM stock solution of dNTP. So I'm assuming you just combine all four at equal amounts to create a 100 mM solution of dNTP. Then you dilute 1/10 to create a 10 mM stock of dNTP which will be suitable for PCR? Never done it this way, and I'm 99% sure this will work, however in Science, even the little things get you.
That won't work- because if you are adding equal amounts of four 100mM solutions together, you are increasing the total volume by a factor of 4 and therefore diluting each dNTP 1/4.
What I do is add 10ul of each dNTP then make up to 100ul with nuclease free water for a 1/10 dilution.
leelee is right - combining equal amounts you will end up with a 25 mM solution which you need to dilute further to 10 mM adding the 1.5 Volume of water.
look on page 2, this table will help you to understand the way it works (or tell you the solution of your question).....http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_r0181.pdf
leelee on Mon Dec 12 11:37:35 2011 said:
That won't work- because if you are adding equal amounts of four 100mM solutions together, you are increasing the total volume by a factor of 4 and therefore diluting each dNTP 1/4.
What I do is add 10ul of each dNTP then make up to 100ul with nuclease free water for a 1/10 dilution.
Ok. I thought by 10 mM solution of dNTP's I thought this meant of total dNTP's not each individual of the four dNTP's?
You need 10mM of each
10 mM dNTP mix:
1 microliter 100 mM dATP
1 microliter 100 mM dGTP
1 microliter 100 mM dCTP
1 microliter 100 mM dTTP
6 microliter H2O.
Multiply as needed. I usually make 400 microliter stock for myself: 240 microliter water, with 40 microliter of each 100 mM nucleotide.
All above discussed dilutions have mistakes if you carefully look in to the final concentration. So the correct method to make a 10 mM dNTPs is mixing as below,
1 microliter 100 mM dATP
1 microliter 100 mM dGTP
1 microliter 100 mM dCTP
1 microliter 100 mM dTTP
36 microliter H2O
Because, now you have 400 mM dissolved in 40 microliter total volume, 400 mM/40 = 10 mM dNTP mix.
Dilwee on Mon Jul 29 19:51:21 2019 said:
Yes, but not 10mM of each, which is the desired result. Your formula makes it 10mM of total dNTPs.All above discussed dilutions have mistakes if you carefully look in to the final concentration. So the correct method to make a 10 mM dNTPs is mixing as below,
1 microliter 100 mM dATP
1 microliter 100 mM dGTP
1 microliter 100 mM dCTP
1 microliter 100 mM dTTP
36 microliter H2O
Because, now you have 400 mM dissolved in 40 microliter total volume, 400 mM/40 = 10 mM dNTP mix.