DNA Solutions （DNA分离）
互联网
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.
