1. Weigh out 29 g acrylamide and 1 g N,N?/EM>-methylenebisacrylamide.
<CAUTION> Acrylamide is a potent
neurotoxin and is absorbed through the skin. The effects of acrylamide are cumulative. Wear gloves and a mask when weighing powdered
acrylamide and methylenebisacrylamide. Wear gloves when handling solutions containing these chemicals. Although polyacrylamide is
considered to be non-toxic, it should be handled with care because of the possibility that it might contain small quantities of unpolymerized
acrylamide.
2. Add 60 ml distilled water.
3. Heat the solution to 37 C to dissolve the chemicals.
4. Make up volume to 100
ml with distilled water.
<CRITICAL STEP> Cheaper grades of acrylamide and bisacrylamide are often contaminated with metal ions.
Stock solutions of acrylamide can easily be purified by stirring overnight with about 0.2 volume of monobed resin (MB-1, Mallinckrodt),
followed by filtration through Whatman No. 1 paper.
5. Sterilize the solution by filtration through a Nalgene filter (0.45- micron
pore size).
6. Check that the pH of the acrylamide solution is 7.0 or less.
Store the solution in dark bottles at 4 C. During storage,
acrylamide and bisacrylamide are slowly converted to acrylic acid and bisacrylic acid, so solutions that are older than 1 year should
be discarded
40% Acrylamide (for DNA sequencing)(1 liter)
1. Weigh out 380 g acrylamide (DNA-sequencing grade) and 20 g N,N?/EM>-methylenebisacrylamide.
<CAUTION> Acrylamide is a potent neurotoxin and is absorbed through the skin. The effects of acrylamide are cumulative. Wear
gloves and a mask when weighing powdered acrylamide and methylenebisacrylamide. Wear gloves when handling solutions containing these
chemicals. Although polyacrylamide is considered to be non-toxic, it should be handled with care because of the possibility that it
might contain small quantities of unpolymerized acrylamide.
2. Add 600 ml distilled water.
3. Heat the solution to 37 C to dissolve
the chemicals.
4. Make up volume to 1 liter with distilled water.
<CRITICAL STEP> Cheaper grades of acrylamide and bisacrylamide
are often contaminated with metal ions. Stock solutions of acrylamide can easily be purified by stirring overnight with about 0.2
volume of monobed resin (MB-1, Mallinckrodt), followed by filtration through Whatman No. 1 paper.
5. Sterilize the solution by filtration
through a Nalgene filter (0.45- micron pore size).
6. Check that the pH of the acrylamide solution is 7.0 or less.
<STORAGE> Store
the solution in dark bottles at room temperature. During storage, acrylamide and bisacrylamide are slowly converted to acrylic acid
and bisacrylic acid, so solutions that are older than 1 year should be discarded.
0.1 M Adenosine triphosphate (ATP)(1 ml)
1.
Weigh out 60 mg ATP.
2. Add 0.8 ml distilled water.
3. Adjust to pH 7.0 with 0.1 M NaOH.
4. Make up volume to 1 ml with distilled water.
<STORAGE>
Aliquots of appropriate volume should be stored at -70 C.
10 M Ammonium acetate (1 liter)
1. Weigh out 770 g ammonium acetate.
2.
Add 800 ml distilled water to dissolve.
3. Make up volume to 1 liter with distilled water.
4. Sterilize by filtration.
<CRITICAL>
Ammonium acetate is heat labile. Do not autoclave.
<STORAGE> Store at 4 C.
10 % Ammonium persulfate (10 ml)
1. Weigh out
1g ammonium persulfate and dissolve in 8 ml of distilled water
2. Make up volume to 10 ml with distilled water.
<STORAGE> The
solution may be stored for several weeks at 4 C.
1. Weigh out 54 g CaCl2?H2O and dissolve in 170 ml of distilled
water.
2. Make up volume to 200 ml with distilled water.
3. Sterilize the solution by passage through a 0.22-micron filter.
<STORAGE>
Store in 1 ml aliquots at -20 C.
<USAGE> When preparing competent cells, thaw an aliquot and dilute it to 100ml with distilled
water. Sterilize the solution by filtration through a Nalgene filter (0.45 micron), and chill it to 0 C before use.
1. Weigh out 13.5 g CaCl2?H2O and dissolve in 15 ml of distilled water.
2. Make up volume to 20 ml with distilled water.
3.
Sterilize the solution by filtration through a 0.22 micron filter.
<STORAGE> Store in 1 ml aliquots at -20 C.
Deoxyribonucleoside
triphosphates (dNTPs)
1. Dissolve each dNTP in distilled water at an approximate concentration of 100 mM.
2. Adjust to pH 7.0 with
0.05 M Tris base using a micropipette (use pH paper to check the pH).
3. Dilute an aliquot of the neutralized dNTP appropriately,
and read the optical density at the wavelengths given in the table below.
Base
Wavelength Extinction coefficient
(M-1CM-1)
A
259 1.54x104
G
253 1.37x104
C
271
9.1x103
T
260 7.4x103
4.
Calculate the actual concentration of each dNTP. For a cuvette with a path length of 1 cm, absorbance is equal to the product of the
extinction coefficient and the molar concentration.
5. Dilute the solutions with distilled water to a final concentration of 50 mM
dNTP.
<STORAGE> Store each separately at -70 C in aliquots of appropriate volume.
Commonly used solutions (1)--- 1 ) 2 ) 3 ) 4 ) 5 ) 6 )
Commonly used solutions (2)--- 1 ) 2 ) 3 )
Solutions for molecular cloning---1 ) 2 ) 3 ) 4
) 5 ) 6 ) 7 ) 8 ) 9 )
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