Fill-in Deoxynucleotide Preparation: To make 4 ml of the fill-in nucleotides at a concentration of 0.25 mM of each nucleotide, combine the following:

           500 ul PCR dNTPs (2 mM)

            3500 ul ddH2O

Aliquot this into 0.5 ml eppendorftubes with 10 ul in each tube.

To make 4 ml of these nucleotides at a final concentration of 0.25 mM from the stock 100 mM solutions, add the following:

            10 ul   100 mM dATP

           10 ul   100 mM dCTP

            10 ul   100 mM dGTP

           10 ul   100 mM dTTP

            3.6 ml ddH2O

Aliquot into 0.5 eppendorf tubes with 10 ul in each tube.

To order these nucleotides, call Pharmacia at 1 800-526-3593 and use customer number 6933. Order the dNTP set: 27-2035-01 dNTP set (100mM each dATP, dCTP, dGTP, dTTP- each in 250 ul volume) $174.00 for the set.

20% glucose:

                       20 g    d-glucose

                       ddH2O to 100 

                       ml

            filter sterilize

Sterile glycerin (sterile glycerol):

Eitherautoclave the bottle of ACS grade Glycerin (Fisher Scientific #G33-500) with loosened cap after putting autocalve tape on the bottle (to indicate that it has been autoclaved) or we make a 50% solution of the same Glycerin with ultrapure water (which then is easier to work with) and autoclave that in a convenient size bottle.

6 M guanidine HCl, pH 6.4, containing 50 mM Tris-HCl, 20 mM EDTA:

                       573.18 g         guanidine-HCl (Sigma G-4505)

                       50 ml              1 M Tris-HCl, pH 7.6

                       40 ml              0.5 M EDTA, pH 8.0

                       ddH2O to 1 liter

GET/lysozyme solution: 50 mM glucose, 25 mM Tris-HCl, pH 8.0, and 10 mM EDTA, pH 8.0 in double distilled water,

                       0.9 g               d-glucose

                       2.5 ml             1 M Tris-HCl, pH 8.0

                       2 ml                 0.5 M EDTA, pH 8.0

                       ddH2O to 100 ml (filter sterilize and store at 4degC).

Add 2 mg/ml lysozyme (Sigma L-6876) just before use.

1 M HEPES, pH 7.5:

                       23.83 g           HEPES (Sigma H-3375)

                       ddH2O to 100 ml

adjust pH to 7.5 with potassium hydroxide (KOH) (store at 4deg.C).

IPTG (isopropyl b-D-thiogalactopyranoside):25 mg/ml IPTG in double distilled water

                       250 mg           IPTG (Sigma I-5502)

                       ddH2O to 10 ml (aliquot and store at -20degC)

1 M isocitrate (sodium salt-dihydrate):

                       29.41 g           Na3isocitrate-2H2O (Sigma C-7254)

                       ddH2O 

                       to 100 ml

10x Kinase buffer: 500 mM Tris-HCl, pH 7.6, 100 mM MgCl2, and 100 mM DTT in sterile double distilled water.

                       5 ml                 1 M Tris-HCl, pH 7.6

                       1 ml                 1 M MgCl2

                       1 ml                 1 M DTT

                       sddH2O to 10 ml (store in 25 ml aliquots at -20deg.C).

Kanamycin sulfate (Kan): Stock of 5 mg/ml in sterile double distilled water (sddH2O).

                       0.5 g   Kanamycin (Boehringer 

                       Mannheim 106 801)

                       sddH2O to 100 ml (Add to media 

                       for final conc. 20 ug/ml)

1M KCl (potassium chloride):

                       7.5 g               KCl

                       ddH2O to 100 ml

Lambda plates:

                       10 g                Bacto-tryptone (Difco 0123-01-1)

                       15 g                Bacto-agar (Difco 0140-01)

                       2.5 g               NaCl

                       ddH2O to 1 L

autoclave to sterilize and pour into sterile petri dishes (approx. 20 ml/plate).