Cyclic Mixture Mutagenesis \\ for DNA-Based Computing -- Mixture Recipies<A NAME="2"> </A><A NAME="sec:recipies"> </A>

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Mixture Recipies

Taq buffer:

         3.5 ml  dH2O
         5   ml  1M TrisCl, pH 9.0   500 mM
         1.5 ml  1M MgCl2            150 mM
        -------
        10   ml  10X Taq sequencing buffer

EDTA dilution:

        490 ul  dH2O
         10 ul  0.5 M EDTA   10 mM
        ------
        500 ul  10 mM EDTA
 
 
        450 ul  dH2O
         50 ul  10 mM EDTA   1 mM
        ------
        500 ul  1 mM EDTA

dNTP dilution (for each of A, C, G, T):

        495 ul  dH2O
          5 ul  100 mM dNTP   1 mM
        ------
        500 ul  1 mM dNTP
 
 
        450 ul  dH2O
         50 ul  1 mM dNTP   0.1 mM
        ------
        500 ul  0.1 mM dNTP

Extra dilution for dATP:

        450 ul  dH2O
         50 ul  0.1 mM dATP   0.01 mM
        ------
        500 ul  0.01 mM dATP

TrisCl/EDTA:

        160 ul  dH2O
         20 ul  1 M TrisCl, pH 7.5   0.1 M
         20 ul  1 mM EDTA            0.1 mM
        ------
        200 ul  0.1 M TrisCl (pH 7.5)/0.1 mM EDTA

dNTP chase:

        400    ul  dH2O
         50    ul  0.1 M TrisCl (pH 7.5)/  0.01 mM
                   0.1 mM EDTA             0.01 mM \_ 0.1 mM
         45    ul  1 mM EDTA               0.09 mM /
          1.25 ul  100 mM dATP             0.25 mM
          1.25 ul  100 mM dCTP             0.25 mM
          1.25 ul  100 mM dGTP             0.25 mM
          1.25 ul  100 mM dTTP             0.25 mM
        ---------
        500    ul  dNTP chase

Sanger mixes:

         4.2 ul  dH2O
         6   ul  1 mM EDTA     0.1 mM EDTA
        12   ul  .01 mM dATP   2 uM
        12   ul  .1 mM dCTP    20 uM
        12   ul  .1 mM dGTP    20 uM
        12   ul  .1 mM dTTP    20 uM
         1.8 ul  5 mM ddATP    150 uM
        -------
        60   ul  A Taq Sanger Mix
 
 
         6   ul  1 mM EDTA     0.1 mM EDTA
        12   ul  .01 mM dATP   2 uM
        12   ul  .1 mM dCTP    20 uM
        12   ul  .1 mM dGTP    20 uM
        12   ul  .1 mM dTTP    20 uM
         6   ul  5 mM ddCTP    500 uM
        -------
        60   ul  C Taq Sanger Mix
 
 
         3.3 ul  dH2O
         6   ul  1 mM EDTA     0.1 mM EDTA
        12   ul  .01 mM dATP   2 uM
        12   ul  .1 mM dCTP    20 uM
        12   ul  .1 mM dGTP    20 uM
        12   ul  .1 mM dTTP    20 uM
         2.7 ul  5 mM ddATP    225 uM
        -------
        60   ul  G Taq Sanger Mix
 
 
         6   ul  1 mM EDTA     0.1 mM EDTA
        12   ul  .01 mM dATP   2 uM
        12   ul  .1 mM dCTP    20 uM
        12   ul  .1 mM dGTP    20 uM
        12   ul  .1 mM dTTP    20 uM
         6   ul  5 mM ddATP    500 uM (want 520)
        -------
        60   ul  T Taq Sanger Mix

Oligo dilutions:

        44.9 ul  H2O
         1   ul  112 uM O1      2.44 uM
        -------
        45.9 ul  2.44 uM O1
 
 
        36.3 ul  H2O
         1   ul  91 uM O2       2.44 uM
        -------
        37.3 ul  2.44 uM O2
 
 
        39.2 ul  H2O
         1   ul  98 uM O3       2.44 uM
        -------
        40.2 ul  2.44 uM O3

Also prepare (not described) at least 6 ul 0.5 U/ul Taq DNA polymerase in 1X Taq sequencing buffer, iced.

Oligo-template DNA mixes (one for each of the 3 oligos):

         vol.    substance                      final conc
        -------  ---------                      ------------
         3   ul  1 ug/ml (500 nM) KSII          150 ng/ml (75 nM)
         3   ul  2.44 uM primer in H2O          367 nM
         6   ul  10X Taq sequencing buffer      3 X
         6   ul  10 mCi/ml [alpha-S-35]dATP     3 mCi/ml
         2   ul  H2O
        -------
        20   ul  O# DNA mix

Reaction mixes (one for each oligo & Sanger mix combination):

         4.5 ul  O# DNA mix:
                   150 ng/ml KSII       50 ng/ml (25 nM)
                   367 nM primer        122 nM
                   3 X Taq seq buf      1 X
                   3 mCi/ml [a35S]dATP  1 mCi/ml
         9   ul  N Sanger mix           2/3 X
        -------
        13.5 ul  Reaction mix O# N

- Michael P. Frank, September 12, 1995. Formatted using HyperLaTeX-1.3.