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【技术文章】常用试剂, 缓冲液配置方法

发布时间:2020-04-20 阅读次数:39
常用试剂, 缓冲液配置方法*
Tris·Cl, 1 M
Dissolve 121 g Tris base in 800 ml H2O
Adjust to desired pH with concentrated HCl
Mix and add H2O to 1 liter
Approximately 70 ml of HCl is needed to achieve a pH 7.4 solution, and approximately 42ml for a
solution that is pH 8.0.
IMPORTANT NOTE: The pH of Tris buffers changes significantly with temperature,decreasing
approximately 0.028 pH units per 1
°C. Tris-buffered solutions should be adjusted to the desired pH at
the temperature at which they will be used. Because the pK
a of Tris is8.08, Tris should not be used as a
buffer below pH
7.2 or above pH 9.0.
EDTA (ethylenediamine tetraacetic acid), 0.5 M (pH 8.0)
Dissolve 186.1 g Na2EDTA·2H2O in 700 ml H2O
Adjust pH to 8.0 with 10 M NaOH (∼50 ml)
Add H2O to 1 liter
HCl, 1 M
Mix in the following order:
913.8 ml H2O
86.2 ml concentrated HCl
MOPS buffer
0.2 M MOPS [3-(N-morpholino)propanesulfonic acid], pH 7.0
0.5 M sodium acetate
0.01 M EDTA
Store in the dark and discard if it turns yellow.
PBS (phosphate-buffered saline)
10xstock solution, 1 liter:
Working solution, Ph~7.3:
80 g NaCl 137 mM NaCl
2 g KCl 2.7 mM KCl
11.5 g Na2HPO4·7H2O 4.3 mM Na2HPO4·7H2O
2 g KH2PO4 1.4 mM KH2PO4

Potassium acetate buffer, 0.1 M
Solution A: 11.55 ml glacial acetic acid/liter (0.2 M).
Solution B: 19.6 g potassium acetate (KC2H3O2)/liter (0.2 M).
Referring to Table A.2.2 for desired pH, mix the indicated volumes of solutions A
and B, then dilute with H2O to 100 ml.
This may be made as a 5- or 10-fold concentrate by scaling up the amount of potassium acetate in the
same volume. Acetate buffers show concentration-dependent pH changes, so check concentrate pH by

diluting an aliquot to the final concentration. To prepare buffers with pH intermediate between the
points listed in Table A, prepare closest higher pH, then titrate with solution A
Table A Preparation of 0.1 M Sodiumand Potassium Acetate Buffers
Desired
pH
Solution A
(ml)
Solution B
(ml)
3.6 46.3 3.7
3.8 44.0 6.0
4.0 41.0 9.0
4.2 36.8 13.2
4.4 30.5 19.5
4.6 25.5 24.5
4.8 20.0 30.0
5.0 14.8 35.2
5.2 10.5 39.5
5.4 8.8 41.2
5.6 4.8 45.2
Potassium phosphate buffer, 0.1 M
Solution A: 27.2 g KH2PO4 per liter (0.2 M).
Solution B: 34.8 g K2HPO4 per liter (0.2 M).
Referring to Table A.2.3 for desired pH, mix the indicated volumes of solutions A and B, then
dilute with H2O to 200 ml.
This may be made as a 5- or 10-fold concentrate by scaling up the amount of potassium phosphate in
the same volume. Phosphate buffers show concentration-dependent pH changes, so check concentrate
pH by diluting an aliquot to the final concentration
TableB Preparation of 0.1 M Sodium and Potassium Phosphate Buffers
Desired
pH
Solution A
(ml)
Solution B
(ml)
Desired
pH
Solution A
(ml)
Solution B
(ml)
5.7 93.5 6.5 6.9 45.0 55.0
5.8 92.0 8.0 7.0 39.0 61.0
5.9 90.0 10.0 7.1 33.0 67.0
6.0 87.7 12.3 7.2 28.0 72.0
6.1 85.0 15.0 7.3 23.0 77.0
6.2 81.5 18.5 7.4 19.0 81.0
6.3 77.5 22.5 7.5 16.0 84.0
6.4 73.5 26.5 7.6 13.0 87.0
6.5 68.5 31.5 7.7 10.5 90.5
6.6 62.5 37.5 7.8 8.5 91.5
6.7 56.5 43.5 7.9 7.0 93.0
6.8 51.0 49.0 8.0 5.3 94.7
.S
odium acetate buffer, 0.1 M

Solution A: 11.55 ml glacial acetic acid/liter (0.2 M).
Solution B: 27.2 g sodium acetate (NaC2H3O2·3H2O)/liter (0.2 M).
Referring to Table A.2.2 for desired pH, mix the indicated volumes of solutions A
and B, then dilute with H2O to 100 ml. (See Potassium acetate buffer recipe for
further details.)
Sodium phosphate buffer, 0.1 M
Solution A: 27.6 g NaH2PO4·H2O per liter (0.2 M).
Solution B: 53.65 g Na2HPO4·7H2O per liter (0.2 M).
Referring to Table A.2.3 for desired pH, mix the indicated volumes of solutions A
and B, then dilute with H2O to 200 ml. (See Potassium phosphate buffer recipe for
further details.)
TAE (Tris/acetate/EDTA) electrophoresis buffer
50× stock solution:
242 g Tris base
57.1 ml glacial acetic acid
37.2 g Na2EDTA·2H2O
H2O to 1 liter
TBE (Tris/borate/EDTA) electrophoresis buffer
10× stock solution, 1 liter:
108 g Tris base (890 mM)
55 g boric acid (890 mM)
40 ml 0.5 M EDTA, pH 8.0 (see recipe; 20 mM)
TE (Tris/EDTA) buffer
10 mM Tris·Cl, pH 7.4, 7.5, or 8.0 (or other pH; see recipe)
1 mM EDTA, pH 8.0
*:出自 Current Protocols in Molecular Biology [B], Wiley Online Library,2007

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