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Buffers

Buffers

Buffer Formula (required precision; 2 %)

Always to try to prepare buffer solution at the temperature and concentration before planing to use during the experiment. Stock solutions are acceptable as long as pH adjustment made after temperature and concentration adjustment. Also good buffers, e.g., Tris and Phosphate, are stable for a long time period.

10X TE (10:2)

1X 1L 10X
10mM Tris-HCl 8.9 g Tris-HCl
5.3 g Tris-Base
2 mM Na3EDTA 7.4 g
pH 8 at 25 C. Mix and store at 4 C. Periodically dump the 1X TE and make up the fresh 1X TE from 10X stock.

10X TE (10:1)

1X 1L 1X 1L 10X
10mM Tris-HCl 1.06 g Tris-HCl 10.6 g
0.39 g Tris-Base 3.94 g
1mM Na2EDTA 0.475 g 4.75 g
pH 8 at 25 C. Mix and store at 4 C.

10X TE (50:50)

1X 1L 10X
50 mM Tris-HCl 44.4 Tris-HCl
26.5 Tris-Base
50 mM Na3EDTA 186 g
Store at 4 C.

TE (25:10) 50 mM Glucose

1X 0.25 L 1X
25 mM Tris-HCl 0.55 g Tris-HCl
0.33 g Tris-Base
10 mM Na3EDTA 0.92 g
50 mM Glucose 2.25 g
Store at 4 C.

50 mM Tris pH 8.0 10 mM EDTA

50 mM 1 M Tris pH 8.0 5 ml
1 mM 0.5 M EDTA 2 ml
H2O up to 100 ml
Store at 4 C.

1 M Tris pH 8.0, 1.5 M NaCl

1X 1L 1X 2L 1X
1M Tris-HCl 88.8 g Tris-HCl 178 g
53.0 g Tris-Base 106 g
1.5 M NaCl 87.7 g 175 g
Store at 25 C (room temperature is OK)

1 M Tris pH 7.4, 1.5 M NaCl

1X 1L 1X 2L 1X
1M Tris-HCl 132.2 g Tris-HCl 264.4 g
19.4 g Tris-Base 38.8 g
1.5 M NaCl 87.7 g 175 g
Store at 25 C (room temperature is OK)

0.5 M Tris pH 7.5, 1.5 M NaCl

Conc. 1 L 2L 3 L
0.5 M Tris Tris-HCl 63.5 g 127.0 190.0
Tris-Base 11.8 g 23.6 g 35.4 g
1.5 M NaCl 87.6 g 175.0 g 262.8 g
Store at 25 C (room temperature is OK)

10 mM Tris pH 7.5, 15 mM NaCl


Conc. Stock Volume
10 mM 1 M Tris pH 7.5 100 µl
15 mM 5 M NaCl 30 µl
H2O to 10 ml
Store at 4 C.

10X TBE Buffer (pH 8)

1X 1L 10X 4L 10X
89 mM Tris-Base 108 g 432 g
89 mM Boric acid 55 g 220 g
2 mM Na2EDTA 9.3 g 37.2 g
EtBr (10mg/ml) 0.5 ml 2 ml
Mix and store at room temperature without EtBr, 4 C with EtBr in a brown bottle. Use EtBr stock solution (10 mg EtBr/ml) when TBE is made.

50X TAE Buffer (pH 8)

1X 1L 50X 1 L 10X
40 mM Tris-acetate 242 g Tris Base 48.4 g
57.1 ml acetic acid 11.4 ml
2 mM Na2EDTA 37 g 7.4 g
EtBr (10mg/ml) 2.5 ml 0.5 ml
Mix and store at room temperature without EtBr, 4 C with EtBr in a brown bottle. Use EtBr stock solution (10 mg EtBr/ml) when TAE is made.

Cotton DNA Extraction buffer (pH 6)

Conc. Stock 1L 1X
100 mM Na2citrate.2H2O 29.4 g
Glucose 63 g
5 mM 0.5 M Na2EDTA 10 ml
Na2Diethyldithiocarbamic acid 10 g
PVP-40,000 MW 20 g
BSA 10 g
Adjust to pH 6.0 with HCl. Store at 4 C.

RSB (Reaction Stop Buffer)

Conc. Stock Volume (ml)
10 mM 1 M Tris pH 8.0 1.0
2 mM 0.5 M EDTA 0.4
0.2 % 20 % SDA 1.0
H2O 97.6
Store at room temperature.

STE (Sodium chloride and TE) pH 8.0

Conc. Stock 1X 1 L 10X 1L
10 mM 1M Tris pH 8.0 10.0 8.88 g Tris-HCl
5.3 g Tris-Base
1 mM 0.5 M EDTA 2.0 4.65 g Na2EDTA
100 mM 5 M NaCl 20.0 5.84 g NaCl
H2O up to 1 L up to 1 L
pH 8.0 at 25 C. Store at 4 C.

1.5 M NaCl 0.5 N NaOH

1X 1L 1X 2L 1X
0.5 N NaOH 20.0 g 40 g
1.5 N NaCl 87.7 g 175 g
Store at room temperature.

0.2 N NaOH 0.6 M NaCl

Conc. 1L 2L 3L 4L
0.2 N NaOH 8.0 g 16.0 g 24.0 g 32.0 g
0.6 M NaCl 35.04 g 70.08 g 105.2 g 140.2 g
Store at room temperature.

20X SSC (Adjust pH 7.0)

1X 1L 20X 2L 4L 6L
150 mM NaCl 175.3 g 350.6 g 701 g 1052 g
15 mM Na3Citrate 88.3 g 176.6 g 353 g 530 g
Mix, adjust pH to 7.0 with HCl, and store at 4 C

25X SSC (Adjust pH 7.4)

25X 1L 4L
3.0 M NaCl 219 g 876 g
0.3 M Na2Citrate 110 g 440 g
Mix, adjust pH to 7.4 with HCl, and store at 4C.

3 M K 5 M Ac

1X 200 ml 1 X
3 M KAc 29.4 g (or 20 ml of 5 M KAc)
2 M Acetic acid 23 ml
Dissolve the KAc in 150 ml of H2O, bring to 177 ml, and then add the glacial acetic acid. Mix and store at 4 C.

Minimal Hybridization Buffer

Conc. Stock 1L 2L
10 % 50 % PEG 200 400
7 % 20 % SDS 350 700
0.6 X 25 X SSC 24 48
10 mM 1 M NaHPO4 10 20
5 mM 0.5 M EDTA 10 20
100 µg/ml Denature Salmon Sperm 10 20
H2O 396 792
Aliquote 45 ml into 50 ml PPT, store at -20 C.

Oligo Buffer


After preparing the following stock solutions, mix A, B, and C in a ratio of 1:2.5:1.5, repectively.

Solution A

Stock
2-mercaptoethanol (bME) 18 µl
DXTPs (A, T, G) 5 µl each
Solution O 850 µl
Store at -20 C

Solution O

Conc. Stock 250 ml 100 ml
1.47 M Tris-Base 44.52 g 17.81 g
0.147 M MgCl2 7.47 g 2.99 g
Adjust pH to 8.0 with conc. HCl.

Solution B

: 2M hepes buffer
Conc. Stock 250 ml 100 ml
2 M Hepes 119.15 g 47.66 g
Adjust pH to 6.6 with 4 N NaOH. Store at 4 C.

Solution C

Hexamer or oligo nucleotides. Add 55 µl H2O directly to the Pharmacia bottle which contains 50 units of lyophilized hexamer. Store at -20 C.

Klenow

Dilute to 1 unit Klewnow fragment by adding klewnow buffer. Stock Klenow from BRL comes as 500 units in 84 µl. Dilute to 1 unit by adding 420 µl of klenow buffer to the 500 unit/84 µl stock. Store - 20 C.

Klenow buffer (250 ml)

Conc Stock
7 mM Tris-HCl 0.211 g
7 mM MgCl2 0.355 g
50 mM NaCl 0.730 g
50 % Glycerol 125 ml
Add the above to about 80 ml H2O. Stir to dissolve. Adjust volume to 250 ml with H2O. Store at -20 C.

REact buffer (See Restriction enzyme buffer formula)

10 X REact buffer 2 : for, e.g. Hind III and Sst 1 (5 ml)

1 X Conc. Stock
500 mM 1 M Tris (pH 8.0) 2.5 ml
100 mM 1 M MgCl2 0.5 ml
500 mM NaCl 0.5 ml
25 mM Spermidine(3HCl) 0.032 g
H2O 1.5 ml
Mix, aliquote 2 ml in screw-cap vial, store at -20 C.

10 X REact buffer 3 : for, e.g. Eco RI (5.0 ml)

1 X Conc Stock
500 mM 1M Tris (pH 8.0) 2.5 ml
100 mM 1M MgCl2 0.5 ml
1 M 5 M NaCl 1.0 ml
25 mM Spermidine(3HCl) 0.032 g
H2O 1.0 ml
Mix, aliquote 2 ml in screw-cap vial, store at -20 C.