Table of Contents

10X TBE

40% Acrylamide Stock

Alkaline lysis solution

10X TBE:

216 g Tris base

110 g boric acid

16.6 g EDTA

Add water to 2 liters.

40% Acrylamide/Bisacrylamide (40% A&B):

38 g Acrylamide (Kodak 5521)

2 g N,N-Methylene-bisacrylamide (Kodak 8383)

Dissolve in approx. 80 ml of double distilled water and then deionize by stirring with 5 g of Amberlite MB-1 (Sigma MB-1A) for 1 hour at room temperature. Suction filter to remove the Amberlite and adjust to a final volume of 100 ml with double distilled water. (store at 4deg.C).

10x Agarose gel loading dye:

15% Ficoll, 0.2% bromophenol blue, 0.2% xylene cyanol FF in double distilled water.

1.5 g Ficoll (Sigma F-2637)

0.02 g Bromophenol blue (Sigma B-0126)

0.02 g xylene cyanole FF (Kodak T-1579)

ddH₂ O to 10 ml (store at -20deg.C).

Alkaline lysis solution (NaOH/SDS) :

0.2 N NaOH, 1% SDS in ddwater.

20 ml of 1 N NaOH (or 0.8 gms)

10 ml of 10% SDS (or 1.0 gms)

ddH₂ O to 100 ml (make fresh)

15% Ammonium persulfate (APS):

1.5 g APS (Kodak 11151)

ddH₂ O to 10 ml (store at 4deg.C).

100 mM rATP (adenosine triphosphate) :

619 mg dipotassium ATP (ICN 100004)

sddH₂ O to 10 ml (aliquot and store at -20℃).

1 mg/ml BSA (bovine serum albumin) :

5 mg BSA (Sigma A-9647)

sddH₂ O to 5 ml (aliquot and store at -20℃)

100 mM calcium chloride (CaCl₂) :

1.48 g CaCl₂-2H₂ O

ddH₂ O to 100 ml

autoclave to sterilize (store at 4deg.C).

50 mM calcium chloride :

0.74 g CaCl₂-2H₂ O

ddH₂ O to 100 ml

autoclave to sterilize (store at 4deg.C).

Deionized formamide :

Stir formamide (Schwarz/Mann Biotech 800686) with Amberlite MB resin, 10 g. per 100 ml, for one hour to deionize; filter through Whatman 3MM paper, store in a dark bottle at room temperature or 4deg.C.

10X denaturing buffer : 200 mM Tris-HCl, pH 9.5, 1 mM EDTA, 10 mM spermidine in double distilled water.

2 ml 1 M Tris-HCl, pH 9.5

20 μl 0.5 M EDTA, pH 8.0

1 ml 100 mM spermidine

ddH₂ O to 10 ml (aliquot and store at -20℃)

Diatomaceous earth (100 mg/ml): Suspend 10 g of diatomaceous earth (Sigma D-5384) in 100 ml of distilled water in a 100 ml graduated cylinder, and let it settle down for 3 hours. Decant the supernatant, and resuspend the pellet in 100 ml of 6 M guanidine HCl, pH 6.4, containing 50 mM Tris-HCl, 20 mM EDTA.

Diatomaceous earth-wash buffer : 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0, 50% ethanol in double distilled water.

10 ml 1 M Tris-HCl, pH 8.0

2 ml 0.5 M EDTA, pH 8.0

500 ml 100% ethanol (McCormick Distilling Co., Inc.)

ddH₂ O to 1 L

1 M DTT (Dithiothreitol, Cleland's reagent):

1.54 g DTT (Calbiochem 233155)

ddH₂ O to 10 ml (aliquot and store at -20℃).

DNase-free RNase A

20 mg/ml RNase A in 1 mM NaOAc, pH 4.5.

200 mg RNase A (Sigma R-5500)

3.3 μl 3 M NaOAc, pH 4.5

ddH₂ O to 10 ml

boil for 10 minutes (aliquot and store at -20℃).

0.5 M EDTA , pH 8.0 (disodium ethylenediamine tetraacetate):

186.1 g Na₂EDTA

Dissolve in approx. 400 ml ddH₂ O, adjust pH to 8.0 with 10 N NaOH, and adjust to 1 liter final volume with distilled water

100 mM EDTA :

20 ml 0.5 M EDTA

80 ml ddH₂ O

100 ml

95% ethanol/0.12 M NaOAc (ethanol/acetate):

95 ml 100% ethanol

4 ml 3 M NaOAc pH 4.5

1 ml ddH₂ O

100 ml

5 mg/ml ethidium bromide (EtBr):

500 mg EtBr (Sigma E-8751)

ddH₂ O to 100 ml

FE (formamide/EDTA): 5:1 (v/v) formamide:50 mM EDTA

10 μl ddH₂ O

10 μl 100 mM EDTA

100 μl deionized formamide

make fresh

10X Fill-in/Kinase buffer :

(500 mM Tris-HCl, pH 7.6, 100 mM MgCl2, 10 mM DTT, and 50 ug/ml BSA in double distilled water)

5 ml 1 M Tris-HCl, pH 7.6

1 ml 1 M MgCl₂

100 μl 1 M DTT

500 μl 1 mg/ml BSA

3.4 ml ddH₂ O

10 ml

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 μl PCR dNTPs (2 mM)

3500 μl ddH₂ O

Aliquot this into 0.5 ml eppendorf tubes with 10 μl 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 μl 100 mM dATP

10 μl 100 mM dCTP

10 μl 100 mM dGTP

10 μl 100 mM dTTP

3.6 ml ddH₂ O

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

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

20% glucose:

20 g d-glucose

ddH₂ O to 100 ml

filter sterilize

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

ddH₂ O 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

ddH₂ O 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)

ddH₂ O 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)

ddH₂ O to 10 ml (aliquot and store at -20℃)

1 M isocitrate (sodium salt-dihydrate) :

29.41 g Na₃isocitrate-2H₂ O (Sigma C-7254)

ddH₂ O 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

sddH₂ O to 10 ml (store in 25 ml aliquots at -20deg.C).

Kanamycin sulfate (Kan): Stock of 5 mg/ml in sterile double distilled water (sddH₂ O).

0.5 g Kanamycin (Boehringer Mannheim 106 801)

sddH₂ O to 100 ml (Add to media for final conc. 20 ug/ml)

1M KCl (potassium chloride):

7.5 g KCl

ddH₂ O to 100 ml

10x Ligation buffer : 50 mM Tris-HCl, pH 7.6, 10 mM MgCl₂, 10 mM DTT, 10 mM rATP, and 250 ug/ml bovine serum albumin (BSA) in sterile double distilled water.

5 ml 1 M Tris-HCl, pH 7.6

1 ml 1 M MgCl₂

1 ml 1 M DTT

1 ml 100 mM rATP

2.5 mg BSA

sddH₂ O to 10 ml (store in 25 ml aliquots at -20deg.C)

Loading dye: 0.3% xylene cyanole FF, 0.3% bromophenol blue, 10 mM EDTA in deionized formamide.

3 g xylene cyanole FF

3 g bromophenol blue

0.2 ml 0.5 M EDTA

deionized formamide to 10 ml

Lysozyme solution : 50 mM Tris-HCl, pH 8.0, 10 mM EDTA, and 5 mg/ml lysozyme in sterile double distilled water.

5 ml 1 M Tris-HCl, pH 8.0

2 ml 0.5 M EDTA

0.5 g lysozyme (Sigma L-6876)

sddH₂ O to 100 ml (make fresh)

1 M MgCl₂ (magnesium chloride):

20.33 g MgCl₂-6H₂ O

ddH₂ O to 100 ml

1 M MgSO₄ (magnesium sulfate):

12.04 g MgSO₄

ddH₂ O to 100 ml (autoclave)

1 M MnCl₂ (manganese chloride):

1.98 g MnCl₂ (Sigma M-8530)

ddH₂ O to 10 ml (store protected from light)

1 M MOPS:

20.93 g MOPS (Sigma M-1254)

Dissolve in 80 ml ddH₂ O, adjust pH to 7.5 with 1 N NaOH, and bring volume to 100 ml.

10X MOPS buffer : 400 mM MOPS, pH 7.5, 500 mM NaCl, 100 mM MgCl₂ in double distilled water.

400 μl 1 M MOPS, pH 7.5

170 μl 3 M NaCl

100 μl 1 M MgCl₂

330 μl ddH₂ O

1 ml

2.7 M MOPS (acid form):

5.65 g MOPS (acid form)

ddH₂ O to 10 ml

MOPS-Acid buffer: 1.35 M MOPS (acid form), 100 mM MgCl² in double distilled water.

500 μl 2.7 M MOPS (acid form)

100 μl 1 M MgCl₂

400 μl ddH₂ O

1 ml

10X Mn²⁺/isocitrate buffer : 50 mM MnCl₂, 150 mM isocitrate (Na salt), 25% glycerol in double distilled water

50 μl 1 M MnCl₂

150 μl 1 M isocitrate

250 μl glycerol

550 μl ddH₂ O

1 ml

10x MTBE (Modified Tris-borate-EDTA buffer): 1.3 M Tris, 0.4 M Boric acid, and 0.02 M EDTA in double distilled water.

162 g Tris base

27.5 g Boric acid

9.3 g Na₂EDTA

ddH₂ O to 1 L

Nucleotide ordering information:

100 mM dATP 27-2050-01 Pharmacia

100 mM dCTP 27-2060-01 Pharmacia

100 mM dGTP 27-2070-01 Pharmacia

10 mM c7dGTP 988 537 Boehringer-Mannheim

100 mM dTTP 27-2080-01 Pharmacia

5 mM ddATP 27-2057-00 Pharmacia

5 mM ddCTP 27-2065-00 Pharmacia

5 mM ddGTP 27-2075-00 Pharmacia

5 mM ddTTP 27-2085-00 Pharmacia

20 mM dNTP stocks : Prepare from 100 mM stocks

80 μl 100 mM dNTP

40 μl 50:1 TE buffer

280 μl ddH₂ O

400 μl

5 mM dNTP stocks : Prepare from 20 mM stocks

25 μl 20 mM dNTP

10 μl 50:1 TE buffer

65 μl ddH₂ O

100 μl

2 mM dNTPs : 2 mM dATP, dCTP, dGTP, and dTTP in 5 mM Tris-HCl, pH 7.6, 0.1 mM EDTA

100 μl 20 mM dATP

100 μl 20 mM dCTP

100 μl 20 mM dGTP

100 μl 20 mM dTTP

100 μl 50:1 TE buffer

500 μl ddH₂ O

1 ml

2 mM [alpha]-S-dNTPs : 2 mM [alpha]-S-dATP, [alpha]-S-dCTP, [alpha]-S-dGTP, and [alpha]-S-dTTP in 5 mM Tris-HCl, pH 7.6, 0.1 mM EDTA

100 μl 20 mM [alpha]-S-dATP

100 μl 20 mM [alpha]-S-dCTP

100 μl 20 mM [alpha]-S-dGTP

100 μl 20 mM [alpha]-S-dTTP

100 μl 50:1 TE buffer

500 μl ddH₂ O

1 ml

3M NaCl (sodium chloride):

17.53 g NaCl

ddH₂ O to 100 ml

10N NaOH (sodium hydroxide):

40 g NaOH

ddH₂ O to 100 ml.

1N NaOH :

10 ml 10 N NaOH

ddH₂ O to 100 ml

9.5M NH₄OAc (ammonium acetate):

73.23 g NH₄OAc

ddH₂ O to 100 ml

8.0M NH₄OAc :

61.69 g NH4OAc

ddH₂ O to 100 ml

10X PCR buffer : 500 mM KCl, 100 mM Tris-HCl, pH 8.5, 15 mM MgCl₂ in sterile double distilled water

5 ml 1 M KCl

1 ml 1 M Tris-HCl, pH 8.5

150 μl 1 M MgCl₂

ddH₂ O to 10 ml

PCR Deoxynucleotide Preparation : To make 12.5 ml of the PCR nucleotides at a concentration of 2 mM each nucleotide, combine the following:

250 μl 100 mM dATP

250 μl 100 mM dCTP

250 μl 100 mM dGTP

250 μl 100 mM dTTP

11.5 ml ddH₂ O

Aliquot this into 25 tubes with 500 μl in each tube. This will keep the nucleotides from being frozed and thawed too much.

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

20% PEG/2.5 M NaCl:

7.3 g NaCl

10 g PEG (MW=8000) (Fisher P156-3)

Dissolve in 40 ml double distilled water by stirring, and then adjust the volume to 50 ml.

50% PEG/0.5 M NaCl :

5.85 g NaCl

100 g PEG (MW=8000) (Fisher P156-3)

Dissolve in 100 ml double distilled water by stirring, and then adjust the volume to 200 ml.

PEG:TE rinse solution : 1:3 solution of 20% PEG containing 2.5M NaCl and 10 mM Tris-HCl, pH 8.0 containing 1 mM EDTA in double distilled water.

250 μl 1 M Tris-HCl, pH 8.0

50 μl 0.5 M EDTA

12.5 ml 20% PEG/2.5 M NaCl.

ddH₂ O to 37.5 ml

Phenol, TE-saturated: add an equal volume of 10 mM Tris-HCl, pH 7.5-8.0, 1 mM Na₂EDTA to ultrapure phenol, mix well, allow phases to separate, remove and discard upper (aqueous) phase. Repeat until the pH of the aqueous phase is between 7.5-8.0 (store at 4℃).

Phenol/chloroform/isoamyl alcohol (25:25:1):

100 ml TE-saturated phenol

100 ml chloroform

4 ml isoamyl alcohol

204 ml

2M NaOAc (sodium acetate) :

27.22 g NaOAc-3H₂ O

ddH₂ O to 100 ml

3M NaOAc , pH 4.5:

408.24 g NaOAc-3H₂ O

Dissolve in approx. 800 ml ddH₂ O , adjust pH to 4.8 with glacial acetic acid and bring to a final volume of 1 L with ddH₂ O.

10X Low Salt Restriction enzyme assay buffer : 100 mM Tris-HCl, pH 7.6, 100 mM MgCl₂, and 10 mM DTT in sterile double distilled water.

1 ml 1 M Tris-HCl, pH 7.6

1 ml 1 M MgCl₂

0.1 ml 1 M DTT

ddH₂ O to 10 ml

10X Medium Salt Restriction enzyme assay buffer : 500 mM NaCl, 100 mM Tris-HCl, pH 7.6, 100 mM MgCl₂, and 10 mM DTT in sterile double distilled water.

1.7 ml 3 M NaCl

1 ml 1 M Tris-HCl, pH 7.6

1 ml 1 M MgCl₂

0.1 ml 1 M DTT

ddH₂ O to 10 ml

10X High Salt Restriction enzyme assay buffer : 1M NaCl, 500 mM Tris-HCl, pH 7.6, 100 mM MgCl₂, and 10 mM DTT in sterile double distilled water.

3.3 ml 3 M NaCl

5 ml 1 M Tris-HCl, pH 7.6

1 ml 1 M MgCl2

0.1 ml 1 M DTT

ddH₂ O to 10 ml

10X SmaI Restriction enzyme assay buffer : 200 mM KCl, 100 mM Tris-HCl, pH 7.6, 100 mM MgCl₂, and 10 mM DTT in sterile double distilled water.

2 ml 1 M KCl

1 ml 1 M Tris-HCl, pH 7.6

1 ml 1 M MgCl₂

0.1 ml 1 M DTT

ddH₂ O to 10 ml

RNase T1 : 100 U/ul in 50 mM Tris-HCl, pH 7.6

100 μl RNase T1 (Sigma R-8251) (100,000 U/0.2 ml)

25 μl 1 M Tris-HCl, pH 7.6

375 μl ddH₂ O

500 μl

10% SDS (sodium dodecyl sulfate):

10 g SDS (Fisher S529-3)

ddH₂ O to 100 ml

1X STB buffer : 25% sucrose and 50 mM Tris-HCl, pH 8.0 in double distilled water.

25 g sucrose

5 ml 1 M Tris-HCl, pH 8.0

ddH₂ O to 100 ml (filter sterilize and store at 4degC)

Silanizing reagent : 5% solution of dichloro dimethyl silane in 1,1,1-trichloroethane.

20X SSC (standard saline-citrate):

17.53 g NaCl

8.82 g sodium citrate

Dissolve in approx. 80 ml ddH₂ O, adjust pH to 7.0 with hydrochloric acid (HCl) and bring final volume to 100 ml.

1X SSC (standard saline-citrate):

5 ml 20X SSC

95 ml ddH₂ O

100 ml

5X Taq reaction buffer : 400 mM Tris-HCl, pH 9.0, 100 mM ammonium sulfate ((NH₄)₂SO₄), pH 9.0, 25 mM MgCl2, and 5% dimethyl sulfoxide (DMSO) in sterile double distilled water.

16 ml 1 M Tris-HCl, pH 9.0

4 ml 1 M (NH₄)₂SO₄, pH 9.0

1 ml 1 M MgCl₂

2 ml DMSO

17 ml ddH₂ O

40 ml

5X Taq dilution buffer : 400 mM Tris-HCl, pH 9.0, 100 mM (NH₄)₂SO₄, pH 9.0, and 25 mM MgCl₂ in sterile double distilled water.

16 ml 1 M Tris-HCl, pH 9.0

4 ml 1 M (NH4)2SO4, pH 9.0

1 ml 1 M MgCl₂

19 ml ddH₂ O

40 ml

5X Taq "A" termination mix: 62.5 uM dATP, 250 uM dCTP, 375 uM c7dGTP, 250 uM dTTP and 1.5 mM ddATP in 5 mM Tris-HCl, pH 7.6 and 0.1 mM EDTA.

20 μl 20 mM dATP

80 μl 20 mM dCTP

240 μl 10 mM 7deaza-dGTP

80 μl 20 mM dTTP

1920 μl 5 mM ddATP

640 μl 50:1 TE buffer

3420 μl sddH₂ O

6.4 ml

5X Taq "C" termination mix : 250 uM dATP, 62.5 uM dCTP, 375 uM c7dGTP, 250 uM dTTP and 0.75 mM ddATP in 5 mM Tris-HCl, pH 7.6 and 0.1 mM EDTA.

80 μl 20 mM dATP

20 μl 20 mM dCTP

240 μl 10 mM 7deaza-dGTP

80 μl 20 mM dTTP

960 μl 5 mM ddCTP

640 μl 50:1 TE buffer

4380 μl sddH₂ O

6.4 ml

5X Taq "G" termination mix : 250 uM dATP, 250 uM dCTP, 94 uM c7dGTP, 250 uM dTTP and 0.125 mM ddGTP in 5 mM Tris-HCl, pH 7.6 and 0.1 mM EDTA.

160 μl 20 mM dATP

160 μl 20 mM dCTP

120 μl 10 mM 7deaza-dGTP

160 μl 20 mM dTTP

320 μl 5 mM ddGTP

1280 μl 50:1 TE buffer

10600 μl sddH₂ O

12.8 ml

5X Taq "T" termination mix: 250 uM dATP, 250 uM dCTP, 375 uM c7dGTP, 62.5 uM dTTP and 1.25 mM ddTTP in 5 mM Tris-HCl, pH 7.6 and 0.1 mM EDTA.

160 μl 20 mM dATP

160 μl 20 mM dCTP

480 μl 10 mM 7deaza-dGTP

40 μl 20 mM dTTP

3200 μl 5 mM ddTTP

1280 μl 50:1 TE buffer

7480 μl sddH₂ O

12.8 ml

20X TAE buffer : 0.8 M Tris, 0.4 M NaOAc, and 0.04 M Na2EDTA, and glacial acetic acid to pH 8.3 in double distilled water.

96.9 g Tris base

32.8 g NaOAc-3H₂ O

14.9 g Na₂EDTA

Dissolve in approx. 700 ml of double distilled water, adjust the pH to 8.3 with glacial acetic acid, and bring to 1 L with ddH₂ O.