76 g acrylamide
4 g methylene bisacrylamide
Make up to 200 ml with DDH2O. Store in dark at 4°. (Wrap bottle with tin foil.)
Amp Plates
5 g NaCl
10 g Bactotryptone
5 g yeast extract
15 g agar
DDH2O to 1 liter
Autoclave
Add 0.3 ml amp stock just before pouring.
Ampicillin
Stock = 250 mg/ml
1 g ampicillin sodium (pharmaceutical grade) in 4 ml sterile DDH2O
Store in aliquots at -20°
10X BU Buffer
70 g Na2HPO4 or 132 g Na2HPO4 € 7 H2O
30 g KH2PO4
DDH2O to 1 liter
Autoclave
Benton and Davis Solutions
Denat. 0.1 N NaOH 4 g/l or 10 ml of 10N
1.5 M NaCl 87.5 g/l
Neut. 0.2 M Tris pH 7.5 24.2 g/l
pH to 7.5 with HCl = ~25 ml conc HCl per 2 l
Optical Grade CsCl ;Step Grade Sol'n
CsCl TM10
1.3 g /ml 3.1 g 6.9 ml
1.4 g/ml 3.9 g 6.1 ml
1.5 g/ml 4.5g 5.5 ml
1.6 g/ml 5.1 g 4.9 ml
Vortex. Should go into sol'n easily.
50 mM CaCl2
3.7 g CaCl2 € 2 H2O
Dissolve in 500 ml DD H2O.
Autoclave.
Casamino Acids for Sinsheimer's
9.9 g casamino acids in 837 ml DDH2O
Autoclave.
(Use 1 liter Bellco bottles.)
Chloramphenicol Stock - 45 mg/ml
9 g chloramphenicol
200 ml EtOH
Store at -20°C. Use 3.5 ml/liter.
20 mM DTT
40 µl 1 M DTT stock sol'n
1960 µl chilled sterile water
Aliquots of 100 µl/Eppendorf tube. Freeze.
Dialysis Bags
sodium carbonate 400g (10% )
EDTA 74.4 g (50 mM )
H2O to 4 liters
boil 4X
250 mM EDTA; pH 8.0
186.1 g Na2 EDTA € 2 H2O
Dissolve in ~1700 ml DD H2O. Adjust pH with 50% NaOH to pH 8.0 (~28 ml 50%
NaOH). EDTA won't dissolve until the pH is increased. Make up to 2 liters with DD H2O.
Autoclave.
Ficoll/Bromphenol Blue Loading Buffer
6 g Ficoll
43 g H2O
Heat with stirring.
Add pinch of Bromphenol blue
5 ml 250 mM EDTA
Fly Egg Laying Medium;; (for 18 trays 5" x 5")
2225 ml H2O
88 g agar
360 ml molasses
Autoclave 30 min.
Add 0.5 ml conc. Tegosept (16 g/150 ml 95% EtOH)
20 ml of ethylacetate, after autoclaving.
Freezing Medium ;(2X)
3.0 g NaCl
12.6 g K2HPO4 (anhydrous)
0.9 g NaCitrate € 2 H2O
0.18 g MgSO4 €7 H2O or 0.09 g MgSO4
1.8 g (NH4)2SO4
3.6 g KH2PO4 (anhydrous)
88 g (70 ml) glycerol (glycerin)
Dissolve and adjust volume to 1 liter. Autoclave.
Gelatin; - 10 mg/ml or 1%
2 g gelatin
200 ml DD H2O
Heat and stir to dissolve
Autoclave.
10% Glucose
200 g glucose
Dissolve in 2 liters DD H2O
Autoclave.
Hot Fudge Sauce
1/2 cup butter dash of salt
3 squares baking chocolate 1 cup light cream or evaporated milk
1-1/2 cups sugar 2 teaspoons vanilla
1/2 cup cocoa
Melt butter and chocolate over low heat. Mix in sugar, cocoa and salt until smooth. Add
cream and bring to a boil, stirring constantly. Remove from heat and stir in vanilla. Store at
4°C. Heat before using in microwave or by placing container in a pan of hot water.
Kan Plates
5 g NaCl
10 g Bactotryptone
5 g yeast extract
15 g agar
DD H2O to 1 liter
Add 1 ml Kanamycin stock before pouring.
Kan Amp Plates
As for Kan plates, but add 0.4 ml amp stock.
Kamamycin Stock
Stock: 5 g Kanamycin (Sigma) in 100 ml H2O
Filter sterilize
add 1 ml / liter plates
1 M KCl
37.28 g KCl
Dissolve in 500 ml DDH2O
Autoclave.
5 M KOAc
49 g / 100ml H2O
L Broth
5 g NaCl 10 20
10 g bactotryptone 20 40
5 g yeast extract 10 20
_____________ __ __
1 liter H2O 2 liter 4 liter
Autoclave.
Lambda Plates
Make up in 4 liter flasks. Autoclave 75 minutes with stir bar. Stir while cooling.
1 l 2.5 l
NaCl 5 g 12.5 g
Bacto-Tryptone 10 g 25 g
Yeast extract 5 g 12.5 g
Agar 11 g 28 g
MgSO4 1.2 g 3 g
water to: 1 liter 2.5 liter
Lysis Mixture
500 ml 250 mM EDTA, pH 8
100 ml 1 M Tris, pH 8
2 ml Triton X-100
Dilute to 2 liters with DD H2O.
N.B. Adjust the volume before mixing, due to sudsing.
Autoclave. This will be cloudy after autoclaving but will clear up as it cools.
20% maltose
400 g maltose
Dissolve in 2 liters DDH2O.
Autoclave.
Marker Stock
4 µg l Hind III
5 µg FX Hae III
TE to 400 µl
200 µl Ficoll-BPB with 25 mM EDTA
1 M MgCl2
101.6 g MgCl2 € 6 H2O
Dissolve in 500 ml DDH2O.
Autoclave.
0.1 M MgSO4
12g MgSO4
DDH2O to 1 liter
aliquots of 100 ml/bottle
Autoclave
Minimal Plates
for 500 ml: Autoclave 7.5g agar in 419 ml DDH2O with stir bar.
Add following sterile solutions:
50 ml 10xM9 phosphate
5 ml 10% NH4Cl
5.5 µl 0.1 M MgSO4
20 ml 10% glucose
1 ml 10 mg/ml thiamine HCl (add when ready to pour)
10X M9 Phosphate
30 g KH2PO4
113g Na2HPO4 € 7 H2O / to 1 liter H2O
Autoclave
M9 Medium
per liter:
100 ml 10X phosphate
10 ml 10% NH4Cl
11 ml 0.1 M MgSO4
40 ml 10% glucose
2 ml 10 mg/ml thiamine HCl
837 ml M9 CAA
5 M NaCl;
146 g NaCl
to 500 ml with DD H2O
Autoclave.
(Note: heat to dissolve salt as concentration is near saturation.)
10% NH4Cl
200 grams NH4Cl
DD H2O to 2 liters
Autoclave.
4X Nick Translation Buffer
84 µl 1 M Tris pH 8
336 µl 1 M Tris pH 7.5
42 µl 1 M MgCl2
40 µg Pentex BSA
1498 µl H2O.
O/N Broth
20g Tryptone
10g NaCl
Yeast extract 10g
DD H2O to 1 liter
Autoclave.
add maltose to 0.1% before use if desired (0.5 ml 20% maltose/100 ml O/N).
10% SDS
100 g SDS (Biorad)
DD H2O to 1 liter
DO NOT AUTOCLAVE
20X SSC
350.6 g NaCl
176.5 g NaCitrate
Dissolve in 2 liters DD H2O.
Autoclave.
20X SSCP
800 ml 1M sodium phosphate pH 6.8
176.4g NaCitrate
280.5g NaCl
to 2 liters with DD H2O.
Autoclave.
Sinsheimer's Medium
1.5 ml 5 M NaCl
9.0 ml 10% NH4Cl
36 ml 10% glucose
20 ml 20% maltose
5 ml 1 M MgCl2
2 ml 50 mM CaCl2
840 ml Sinsheimer CAA
3 M sodium acetate
246.1 g sodium acetate (anhydrous)/1 liter H2O.
Autoclave
1 M sodium phosphate pH 6.8
138g NaH2PO4 € H2O
268g Na2HPO4 € 7 H2O
Adjust to 2 liters with DD H2O.
Autoclave.
Southern Blot Sol'ns
4 liters
Denat. 0.5 M NaOH 200 ml 10N NaOH
1.5 M NaCl 350.6 g NaCl
Neut. 1 M Tris pH 7.4 484.4 g Tris pH 7.4
3M NaCl 701.3 g NaCl
pH with >250 ml conc HCl
Stabs
Bactotryptone 8g
NaCl 5g
Difco
agar 6g
Yeast extract 1g
DDH2O to 1 liter
Adjust pH to 7.2-7.4 with about 4 drops of 6 M NaOH per liter. Autoclave 5 min to dissolve
agar. Fill vials with 3 ml each. Autoclave. Leave upright.
25% Sucrose (w/v) in 50 mM Tris HCl, pH 7.5
500g sucrose
100 ml 1 M Tris HCl, pH 7.5
DD H2O to 2 liters
Autoclave.
20X TBE (Tris-borate,; electrophoresis buffer;)
432 g Tris base
220 g Boric acid (crystal dissolves easier than powder)
40.8 g Na2 EDTA € 2 H2O
Dissolve in 2 liters DD H2O
Check: pH = 8.35 for a 1:20 dilution
Autoclave. (Use Bellco 1 liter bottles)
TCM
5 ml 1 M Tris HCl, pH 7.5 (10 mM Tris HCl, pH 7.5)
100 ml 50 mM CaCl2 (10 mM CaCl2)
5.0 ml 1 M MgCl2 (10 mM MgCl2)
Dilute to 500 ml with DD H2O.
Autoclave.
TE pH 8.0 (10 mM Tris; 1 mM EDTA)
10 ml 1 M Tris pH 8.0
4 ml 250 mM EDTA, pH 8.0
Adjust volume to 1 liter with DD H2O.
Autoclave.
TE/0.2 M NaCl
11.6 g NaCl
Dissolve in 1 liter TE.
Autoclave.
Tetracycline
5 g tetracycline (tetracycline HCl, pharmaceutical grade)
100 ml 50% Ethanol / 50% sterile DDH2O
Want 25 µg/ml
Add 0.25 ml/500 ml L-broth
Let agar stir a while after adding antibiotic.
TM10 with Gelatin
2.41 g MgSO4
20 ml 1 M Tris pH 7.5
20 ml 1% gelatin (or 0.2g dry gelatin. Heat to dissolve.)
DD H2O to 2 liters.
Autoclave.
TM10 - 10 mM MgSO4 and 10 mM Tris pH 7.5
2.41 g MgSO4
20 ml 1 M Tris pH 7.5
Make up to 2 liters with DD H2O. Autoclave.
Top Agar
5 g NaCl
10 g bactotryptone
5 g yeast extract
1.2 g MgSO4
7 g Agar DD H2O to 1 liter.
This must be heated to almost boiling. Aliquot 50 ml per milk dilution bottle. Autoclave.
Top Agarose
5 g NaCl
10 g bactotryptone
5 g yeast extract
1.2 g MgSO4
1 liter DD H2O
7 g Agarose (low EEO)
This must be heated to almost boiling. Aliquot 50 ml per milk dilution bottle. Autoclave.
300 mM Tris Base
36.34 g Tris base
to1 liter with DD H2O
Autoclave.
1 M Tris pH 8
177.6 g Trizma HCl
106 g Trizma base
Dissolve in 2 liters DD H2O.
Autoclave.
1 M Tris pH 7.5
254 g Trizma HCl
47.20 g Trizma base
Dissolve in 2 liters DD H2O.
Autoclave.
50 mM Tris pH 8
50 mM EDTA pH 8
15% sucrose (w/v)
Tris 50mM,pH8.0, EDTA 50mM, pH8.0, 15% sucrose (w/v);
100 ml of 1 M Tris pH 8
400 ml of 250 mM EDTA
300 g sucrose
DD H2O to 2 liters.
Autoclave.
2X YT Broth
16g bactotryptone
10g yeast extract
10g NaCl
H2O to 1 liter
Autoclave.
1X YT Agar
Top Bottom
8g 8g Bactrotryptone
5 5 yeast extract
5 5 NaCl
7 15 Agar
___ ___
1 liter 1 liter H2O
Autoclave.
Carrier DNA
10 g herring testis DNA (Sigma Type XIV)
DDH2O to 2 liters
Stir until all DNA is no longer opaque. Autoclave. Freeze until use.
100X Denhardt's Solution
20g polyvinylpyrolidone
20g bovine serum albumin (Sigma Type V)
20g ficoll
10ml 0.25M EDTA
H2O to 1 liter
DNase for Nick-Translation
10-4 mg/ml Worthington DNase (DPFF) in 50% glycerol, 1X NT buffer.
Ethidium bromide
500 mg ethidium bromide (Sigma)/100 ml DDH2O
Loening Buffer
4.36 g Tris base (36 mM)
4.14 g NaH2PO4 € H2O (30 mM)
0.336 g Na2EDTA (1 mM)
DDH2O to 1 liter
Nucleotide triphosphates
To 25 mg nucleotide triphosphate add 350µl sterile water. Bring to pH 7 by adding 1N
NaOH (20-50 µl), using pH paper to test after each addition. Make a 1/2000 dilution into a
buffer of the appropriate pH and measure the optical density at the appropriate wavelength.
The final concentration should be about 100mM.
Nucleotide pH of Wavelength Extinction
dilution buffer coefficient
dATP 7 259 nm 15.2 x103
ATP 7 259 15.4 x103
dCTP 2 (0.01 M HCl) 280 13.1 x 103
CTP 2 280 12.8 x 103
dGTP 7 253 13.7 x 103
GTP 7 253 13.7 x 103
dTTP 7 267 9.6 x 103
UTP 7 262 10.0 x103
NOTE: conc. = optical density/extinction coeff. x dilution factor. So for a 100 mM solution of
UTP, diluted 1/2000 in pH 7 buffer, the optical density at 262 nm will be 0.5: 10-1 M = (0.5
O.D. units/10x103 M/O.D.) x (2x103).
Restriction Enzyme Buffers
See Maniatis
10X Reverse Transcription Buffer
for 5 ml:
2.5 ml 1M Tris HCl, pH8.3 (500mM)
0.6 ml 5 M NaCl (600mM)
0.3 ml 1M Mgcl2
12.5 ml 2 M KCl
Make 100µl aliquots. Add 1µl 1M DTT/ 50µl reaction.