Special care must be taken in the storage and handling of EDC (
). The normal volume for covalent binding of one plate is 10 ml, which leaves 400 µl in surplus.Preparation of buffers for the DIAPOPS procedure
1. Introduction
A number of different types of buffers are used in the DIAPOPS procedure. The preparation of these buffers is described below. The products listed are used by Nunc A/S research laboratory, but are only given as examples.
A list of all buffers used in DIAPOPS is provided below. The compositions and components of the buffers are described in detail below the list. The water used has been purified on a Milli-Q PLUS 185 device, which is also fitted with a sterile filter. The water is designated "Milli-Q water". The preparation of solutions used to mix the DIAPOPS buffers is also described.
2. Buffers for covalent binding of solid phase primer
2 a) 1 ng/µl solid phase primer and 10 mM EDC (1-Ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide) in 10 mM 1-Methylimidazole (1-MeIm) (pH 7.0) (10 ml)
Special care must be taken in the storage and handling of EDC ( ). The normal volume for covalent binding of one plate is 10 ml, which leaves 400 µl in surplus.
Mix:
19.17 mg EDC, 191.70 g/mol Final concentration: 10 mM
Sigma, Cat. No. 7750 (
100 µl 1 M MeIm (pH 7) Final concentration: 10 mM
10 µl 1000 ng/µl solid phase primer in TE-buffer
The volume is adjusted to 10 ml with Milli-Q water, and 100 µl is used for each well.
2 a i) 1 M 1-Methylimidazole (1-MeIm) (10 ml)
Mix:
797.1 µl 1-MeIm MW 82,1 g/mol - d = 1,030 kg/l
(Sigma M-8878),
6 ml Milli-Q water
pH is adjusted to 7,0 with 5 N HCl
The volume is adjusted to 10 ml with Milli-Q water. 1-MeIm is strongly corrosive and harmful. Use gloves.
Mix:
200 ml Milli-Q water
239 ml 32% HCl
(32%. Riedel-de Häen Cat. No. 30720)
Add the HCl slowly to the water to avoid boiling.
Adjust the volume to 500 ml with Milli-Q water.
The solution can be stored at RT.
2 a iii) Solid phase primer
The solid phase primer is supplied either in a solution or as a precipitate. It is very important that the solid phase primer is synthesized with a spacer and a phosphate group in the 5'-end ( ). The primer is dissolved in TE-buffer ( ), and 1000 ng/µl is a convenient concentration for a working solution in the solid phase binding. Aliquots should be taken of the primer before freezing ( ).
2 a iv) TE-buffer
TE is prepared by diluting a 10 concentrated stock solution with distilled water before use.
Mix:
10.0 ml 1 M TRIS (pH 8.0) Final concentration: 100 mM
2.0 ml 0.5 M EDTA (pH 8.0) Final concentration: 10 mM
88 ml Milli-Q water
The solution is autoclaved before use and storage.
2 a vi) 1.0 M TRIS (pH 8.0) (100 ml)
Mix:
8.88 g TRIS-HCl, 157.56 g/mol Final concentration: 0.56 M
Sigma Cat. No. T-3253
5.30 g TRIS base 121.1 g/mol, Final concentration: 0.44 M
Sigma Cat. No. T-1503
80 ml Milli-Q water
The pH is adjusted to 8.0 and the volume is adjusted to 100 ml with Milli-Q water. The buffer can be stored at room temperature.
2 a vii) 0.5 M EDTA (pH 8.0) (500 ml)
Mix:
93.06 g EDTA Final concentration: 0.5 M
ultraPURE, GibcoBRL, Cat. No. 15576-028
400 ml Milli-Q water
10 g NaOH pellets
Riedel-de Häen, Cat. No. 30620
The NaOH pellets are added to raise the pH, but the solution must still be adjusted further to 8.0 with 10 N NaOH. The volume is adjusted to 500 ml with Milli-Q water, and the buffer is autoclaved.
Mix:
400.0 g NaOH
Riedel-de Häen, Cat. No. 30620
The volume is adjusted to 1000 ml with Milli-Q water.
2 b) 0.4 M NaOH and 0.25% Tween 20 (1000 ml)
Mix:
16.0 g NaOH pellets Final concentration: 0.4 M
Riedel-de Häen, Cat. No. 30620
2.5 ml Concentrated Tween 20 Final concentration: 0.25%
Riedel-de Häen, Cat. No. 63158
The volume is adjusted to 1000 ml with Milli-Q water, and must be used the same day it is prepared.
3 a) DIAPOPS buffer: 100 mM TRIS-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20
The DIAPOPS buffer is prepared by diluting a 10 stock buffer 10 to 1 with Milli-Q water.
3 a i) 10 DIAPOPS buffer (1000 ml)
Mix:
127.00 g TRIS-HCl (157.56 g/mol) Final concentration: 0.8 M
TRIS-HCl, Sigma, Cat. No. T-3253
23.60 g TRIS base (121.1 g/mol) Final concentration: 0.2 M
TRIS base, Sigma, Cat. No. T-1503
87.66 g NaCl (58.44 g/mol) Final concentration: 1.5 M
NaCl, Riedel-de Häen, Cat. No. 31434
10.0 ml Concentrated Tween 20 Final concentration: 1% (v/v)
Riedel-de Häen, Cat. No. 63158
The pH must be checked and adjusted to 7.5 with 1 M NaOH.
The volume is adjusted to 1000 ml with Milli-Q water. The buffer can be stored at room temperature.
Mix:
40.0 g NaOH
Riedel-de Haën, Cat. No. 30620
The volume is adjusted to 1000 ml with Milli-Q water.
3 b) DIAPOPS buffer with 10 mg/ml BSA (100 ml)
Mix:
100 ml DIAPOPS wash buffer
1.0 g BSA Fraction V Final concentration: 10 mg/ml
Sigma, Cat.No.: A-4503.
The BSA may take some time to dissolve. The buffer must be prepared freshly.
3 c) PCR mix
The concentrations of the single reagents in the PCR can vary from system to system, and optimization is very important ( ). The listed concentrations are only suggestions. In standard liquid phase PCR a ratio of 1:1 between the concentrations of the primers is used. In DIAPOPS it is very important that the primers are present in different concentrations. The primer used as a solid phase primer, called the downstream primer, must be the lower concentration ( ) of the two primers in the liquid phase. The optimal ratio of the two primer concentrations can also vary from one system to another, but a good, general starting ratio is 1:8 ( ).
One PCR with a total volume of 50 µl typically contains the following components:
5 µl 10 PCR buffer (without MgCl2)
3 µl MgCl2 (30 mM. Final concentration: 1.8 mM MgCl2)
8 µl dNTP mix (1.25 mM of each mononucleotide. End conc.: 0.2 mM)
1 µl Upstream primer (25 pmol/µl. Conc. adjusted with TE-buffer)
1 µl Downstream primer (25/8 pmol/µl. Conc. adjusted with TE-buffer)
1 U Taq DNA polymerase (GibcoBRL. Cat. No. 18038-026)
5 µl Template DNA
The volume is adjusted to 50 µl with Milli-Q water.
3 c i) 10 PCR buffer (without MgCl2) (2.5 ml)
Mix:
250 l TRIS-HCl, (pH 8.3), 1M Final concentration: 100 mM
500 l KCl, 2.5 M Final concentration: 500 mM
25 µl Tween 20 Final concentration: 1%
1725 l Milli-Q water
The buffer is aliquoted into 100 µl portions and stored at -20ºC.
3 c ii) 1M TRIS-HCl, (pH 8.3) (100 ml)
Mix:
6.14 g Trizma HCl
Sigma. cat.no. T-3253
7.40 g Trizma Base
Sigma. cat.no. T-1503
80 ml Milli-Q water
The pH is controlled and adjusted to 8.3 with 5 N HCl.
The volume is adjusted to 100 ml with Milli-Q water
Mix:
18.64 g KCl
Riedel-de Haën, Cat. No. 31248.
The volume is adjusted to 100 ml with Milli-Q water.
3 c iv) 30 mM MgCl2 (100 ml)
This buffer is prepared by diluting a 10 stock buffer of 300 mM, 1:10 with distilled water.
Mix:
6.10 g MgCl2, 6 H2O
Riedel-de Haën, Cat. No. 31413
The volume is adjusted to 100 ml with Milli-Q water
3 c vi) dNTP-mix: 1.25 mM of each nucleotide (1600 µl)
Mix:
20 l dATP (100mM)
20 l dTTP (100mM)
20 l dCTP (100mM)
20 l dGTP (100mM)
dNTP-set (Pharmacia, Cat. No. 27-2035-01)
1520 l Milli-Q water
Aliquots of 100 µl are stored at -20ºC.
3 d) DNA template
The template DNA can be a plasmid, a previously amplified PCR product, or genomic DNA. The purity of the DNA is important on order to avoid materials which may inhibit the Taq DNA polymerase ( ). In theory, the concentration of the DNA can be as low as one copy of template DNA in the 5 µl added and still be detected. In practice, the normal detectable concentration is between 50 and 500 copies in the 5 µl added.
3 e) 0.2 M NaOH, 0.1% Tween 20 (1000 ml)
Mix:
8.0 g NaOH Final concentration: 0.2 M
Riedel-de Häen, Cat. No. 30620
1.0 ml Concentrated Tween 20 Final concentration: 0.1%
Riedel-de Häen, Cat. No. 63158
The volume is adjusted to 1000 ml with Milli-Q water
The buffer must be used the same day as it is prepared.
4. Buffers for detection of solid phase product
4 a) 50 nM biotinylated hybridization probe diluted in 5 x SSC, 0.1% Tween 20 and 0.5% blocking reagent (BR) (10 ml)
10 ml hybridization buffer with probe is enough for 12 NucleoLink Strips, which fill one frame and equal one MicroWell® plate. The biotinylated probe is diluted to 50 pmol/µl in TE-buffer.
Mix:
10 µl biotinylated probe (50 pmol/µl)
9990 µl 5 x SSC, 0.1% Tween 20 and 0.5% BR
100 µl of this hybridization mix is added to each NucleoLink well.
4 a i) 5 x SSC, 0.1% Tween 20, and 0.5% Blocking reagent (BR) (500 ml)
Mix:
125 ml 20 x SSC
0.5 ml Concentrated Tween 20
Riedel-de Häen, Cat. No. 63158
2.5 g Blocking Reagent (BR)
Boehringer Mannheim, Cat. No. 1096 176
The volume is adjusted to 500 ml with Milli-Q water. Blocking reagent is dissolved by heating the buffer to 50ºC in a heating incubator for several hours with regular stirring. After heating, the buffer is cooled to room temperature before the pH is adjusted to 7.0. After the pH adjustment, aliquots of 50 ml can be stored at -20ºC.
Mix:
175.3 g NaCl Final concentration: 3.0 M
Riedel-de Häen, Cat. No. 31434
88.2 g NaCitrat Final concentration: 0.3 M
tri-Sodiumcitrate-Dihydrate, MERCK, Cat. No. 6448
800 ml Milli-Q water
pH is adjusted to 7.0 with a few drops of 10 N HCl.
The volume is adjusted to 1000 ml with Milli-Q water, and the buffer is autoclaved.
4 b) 0.5 x SSC, 0.1% Tween 20 (1000 ml)
Mix:
25 ml 20 x SSC
1 ml Concentrated TWEEN 20 ()
Riedel-de Häen, Cat. No. 63158
The volume is adjusted to 1000 ml with Milli-Q water. The buffer can be stored at room temperature.
4 c) AP conjugated streptavidin diluted 1:3000 in DIAPOPS buffer with 0.5% BR (10 ml)
Mix:
3.3 µl AP conjugated streptavidin
Dako, Code No.: D0396
10 ml DIAPOPS buffer with 0.5% BR
The solution must be used immediately after mixing, and 100 µl is added to each well.
4 c i) DIAPOPS buffer with 0.5% BR (500 ml)
Mix:
50 ml 10 DIAPOPS buffer
2.5 g Blocking Reagent (BR)
Boehringer Mannheim, Cat. No. 1096 176
The volume is adjusted to 500 ml with Milli-Q water. Blocking reagent is dissolved by heating the buffer to 50ºC in a heating incubator for several hours with regular stirring. Aliquots of 50 ml can be stored at -20ºC.
4 d) HRP conjugated streptavidin diluted 1:5000 in DIAPOPS buffer with 0.5% BR (10 ml)
Mix:
2.0 µl HRP conjugated streptavidin
Dako, Code No.: P-0397
10 ml DIAPOPS buffer with 0.5% BR
The solution must be used immediately after mixing, and 100 µl is added to each well.
4 e) 1 mM 4-MUP dissolved in 1 M diethanolamine (pH 9.8), 1 mM MgCl2 (1000 ml)
Mix:
95.6 ml Diethanolamine, MW = 105.14 g/mol, d = 1.10 kg/l.
Riedel-de Häen, Cat. No. 15421
800 ml Milli-Q water
pH is adjusted to 9.8 with 10 M NaOH
Then add the following:
0.2033 g MgCl2, 6 H2O MW = 203.30 g/mol
Riedel-de Häen, Cat. No. 31413
0.2562 g 4-Methylumbelliferyl phosphate, (4-MUP), MW = 256.2 g/mol
Sigma, Cat. No. M-8883
The volume is adjusted to 1000 ml with Milli-Q water
Diethanolamine is difficult to pour out of the measuring glass, which must be rinsed many times. Aliquots of 10 ml of the prepared solution are stored at -20ºC.
4 f) PNPP in 1 M diethanolamine (pH 9.8) and 1 mM MgCl2 (1 or 10 mg/ml pNPP, both concentrations can be used) ( )
The pNPP can be purchased as tablets.
For 1 mg/ml mix
15 ml 1 M diethanolamine buffer (ph 9.8) with 1 mM MgCl2
1 tablet pNPP 15 mg/tablet
Sigma, Cat. No. N-2640
For 10 mg/ml mix
15 ml 1M diethanolamine buffer (ph 9.8)
10 tablets pNPP 15 mg/tablet
The pNPP solution is prepared just before use.
Mix:
50 ml 2 M H2SO4
950 ml Milli-Q water
Mix:
56 ml 95-97% H2SO4
Riedel-de Haën. Cat. No. 30743
444 ml Milli-Q water
4 f iii) 1 M diethanolamine (pH 9.8) with 1 mM MgCl2 (1000 ml)
Mix:
95.6 ml Diethanolamin, MW = 105.14 g/mol, d = 1.10 kg/l.
Riedel-de Häen, Cat. No. 15421
800 ml Milli-Q water
pH is adjusted to 9.8 with 10 M NaOH
Then add
0.2033 g MgCl2, 6 H2O MW = 203.30 g/mol
Riedel-de Häen, Cat. No. 31413
The volume is adjusted to 1000 ml with Milli-Q water.
Diethanoamine is difficult to pour out of the measuring glass, which must be rinsed many times. Diethanolamine buffer can be stored at 4ºC.
Mix:
342.29 g K2HPO4, 3 H2O
Roth, Cat. No. 6878.1
The volume is adjusted to 500 ml with Milli-Q water.