Preparation of Basic Reagents for Biomedical Experiments(生物信息站-生物医学实验方法学）

Preparation of Basic Reagents for Biomedical Experiments

1. 50X Tris-acetate (TAE)

242 g. Tris Base
57.1 ml Glacial acetic acid
100 ml 0.5M EDTA (pH 8.0)
1000 ml ddH2O

2. 5X Tris- borate (TBE)

54 g Tris Base (or Sigma 7-9)
27.5 g Boric acid
20 ml of 0.5M EDTA (pH 8.0)
800 ml ddH2O
Stir to get into solution. Bring up to 1 liter with ddH2O. Does not need to be autoclaved. Remove stir bar.

3. 1M Tris-HCl buffer (pH 8.0)

121.1 g. Tris base
800 ml. ddH2O
Adjust the pH to the desired value (8.0) by adding concentrated HCl.

(for pH 7.4, needs about 70 ml HCl, for pH7.6, needs 60 ml HCl, pH8.0, 42ml HCl)

Notes:

If the 1 M solution has a yellow color, discard it and obtain a better quality Tris.

Although many types of electrodes do not accurately measure the pH of Tris solutions, suitable electrodes can be obtained from most manufacturers.

The pH of Tris solutions is temperature-dependent and decreases approximately 0.03 pH units for each 1°C increase in temperature. For example, a 0.05 M solution has pH values of 9.5, 8.9, and 8.6 at 5 °C, 25 °C, and 37 °C respectively.

4. 0.5M EDTA sulution (pH 8.0)

186.1 g. Disodium ethylenediaminetetraacetate·2H2O
800ml ddH2O
Stir vigorously on a magnetic stirrer. Adjust the pH to 8.0 with NaOH (~20 g of NaOH pellets). Aliquot and sterilize by autoclaving.

Note:

The disodium salt of EDTA will not go into solution until the pH of the solution is adjusted to approximately 8.0 by the addition of NaOH.

5. TE Buffer (pH 8.0)

2 ml 1M Tris HCl (pH 8.0) --------(10 mM)
0.4 ml 0.5M EDTA (pH 8.0)------ (1 mM)
200 ml ddH2O

6. 10% SDS

**Sodium Dodecyl Sulfate also known as sodium lauryl sulfate
100 g. SDS (eletrophoresis-grade)
900 ml ddH2O
----Mix and heat to 68 °C to dissolve. Adjust the pH to 7.02 by adding a few drops of concentrated HCl. Adjust the volume to l liter with ddH2O. Aliquot into containers.

Note:

Wear a mask when weighing SDS and wipe down the weighing area and balance after use, because the fine crystals of SDS disperse easily. Does not need autoclaving.

7. 1X PBS (Phosphate Buffered Saline)

NaCl---------------- -8 g (137 mM )
KCl ----------------0.2 g (2.7 mM )
Na2HPO4----------1.44 g (10 mM)
KH2PO4 ----------0.24 g (2 mM)
H2O-----------------800 ml

Adjust pH of PBS Buffer Solution to 7.4 with HCl. Add water to 1L. 1XPBS can be obtained by dilution of 10XPBS.

8. 50X Denhardt's Solution (for DNA or RNA hybridization)

1% Ficoll 400

1% Polyvinylpyrrolidone

1% Bovine Serum Albumin (Sigma, Fraction V)

H2O to the desired volume

9. 1%Agarose

1 g. agarose
100ml. 0.5x TBE Buffer (or 1x TAE buffer)

Boiling in a microwaver to melt, watch out for spilling.

10. DNA/RNA Sample Loading Buffer

2.5% Dye Stock Solution:

250 mg Bromphenol blue
10 ml ddH20
Mixed in a screw cap glass test tube. Cover with foil. Stored in the VWR refrigerator.

Standard Loading Buffer:

0.25% bromophenol blue (from 2.5% Dye Stock Solution above)
40% (w/v) sucrose in water

Other double dye gel loading buffers:

*Type I 6x Buffer:

0.25% bromophenol blue
0.25% xylene cyanol
40% (w/v) sucrose in ddH2O
---Store at 4 °C.

*Type II 10x Buffer (for 10 ml):

0.25% bromophenol blue ----- (25mg bromophenol blue)
0.25% xylene cyanol
25% Ficoll (type 400) in ddH2O ----- (2.5g Ficoll)
---Store at room temperature.

*Type III 6x Buffer

0.25% bromophenol blue
0.25% xylene cyanol
30% glycerol in ddH2O
---Store at 4 °C.

*Type IV 6x Buffer

0.25% bromophenol blue
40% (w/v) sucrose in ddH2O
---Store at 4 °C.

11. Reagents for Western Blot Analysis

When working with proteins, try to use nanopure water . Preparing the following solutions.

*Homogenizing Buffer (500 ml)
----1190 mg HEPES (pH 7.4)
----93 mg EDTA (or 500 ul of .5M EDTA)
----1 Tablet of Protease Inhibitors
(Cat# 04693159001, Roche Applied Science)
*RIPA Cell Lysis Buffer

----10 mM NaPO4, pH7.2
----0.3 M NaCl
----0.1% SDS
----1% NP40
----1% DOC (deoxycholate)
----2 mM EDTA

*Protease Inhibitors

----Leupeptin 10 mg/mL (1000x, store -20°C otherwise keep on ice)
----Aprotinin 10 mg/mL (1000x, store -20°C otherwise keep on ice)
----PMSF: (phenylmethylsulfonyl flouride, 100x) 200mM in ethanol. Store at 4°C.

**The full RIPA buffer:

20 mM Tris–HCl, pH 7.4,
150mM NaCl,
2 mM EDTA,
1% NP-40,
1% Na deoxycholate,
0.1% SDS,
50mM NaF,
1mM Na3VO4,
1 mM phenylmethylsulfonyl ﬂuoride(PMSF),
10 mg/ml aprotinin
10 mg/ml leupeptin)

*100x Phosphatase Inhibitors (for kinase assay)

----100 mM NaF
----50 mM NaVanadate
----800 mM ß-glycerol phosphate

*5x Laemmli Sample Busser (15 ml)

----1.5 mg SDS
----3.75 mL 1M Tris, pH 6.8
----0.015 mg bromphenol blue
----1.16 mg DTT
----7.5 mL H2O
----add 7.5 mL Glycerol

*10% SDS (see above)

*Buffers for Making Gels
Lower Buffer (for the separating gel):
----Tris 36.4g, adjust pH to 8.8 with 6M HCl------1.5M
----8mL of 10% SDS -----------------------------0.4%
----up to 200mL with water

Stacking (Upper) buffer (for the stacking gel):
----requires fair bit of HCl so start with ~70mL water when add Tris
----Tris 6.06g, adjust pH to 6.8 with 6M HCl------0.5 M
----4 ml of 10% SDS (or 0.4g SDS powder)------0.4%
----up to 100mL with water

For 30% Acrylamid:

*30% Acrylamide:Bis Solution (19:1)

Acrylamide 19 g

N,N'- methylenebisacrylamide (Bis) 1 g

Add distilled H2O to make a final volume of 66.6 ml

*30% Acrylamide:Bis Solution (29:1)

Acrylamide 29 g

N,N'- methylenebisacrylamide (Bis) 1 g

Add distilled H2O to make a final volume of 100 ml

*30% Acrylamide:Bis Solution (37.5:1)

Acrylamide 37.5 g

N,N'- methylenebisacrylamide (Bis) 1 g

Add distilled H2O to make a final volume of 128 ml

If you need 40% acrylamide solutions:

*40% Acrylamide:Bis Solution (19:1)

Acrylamide 38 g

N,N'- methylenebisacrylamide (Bis) 2 g

Add distilled H2O to make a final volume of 100 ml

*40% Acrylamide:Bis Solution (29:1)

Acrylamide 29 g

N,N'- methylenebisacrylamide (Bis) 1 g

Add distilled H2O to make a final volume of 75 ml

*40% Acrylamide:Bis Solution (37.5:1)

Acrylamide 37.5 g

N,N'- methylenebisacrylamide (Bis) 1 g

Add distilled H2O to make a final volume of 96.25 ml

Heat the solution to 37oC to dissolve the chemicals.

Filter the solution through a 0.45 um-membrane filter.

Adjust the pH to less than 7.0.

Store the solution in dark bottles or wrap aluminium foil around bottle (because it is light sensitive) at room temperature for less than 3 months.

*10% APS (Ammonium Persulfate)
----APS 100mg
----H2O 1 ml
Store at -20 °C.

*TEMED

Directly buy from any company (Bio-Rad or Invitrogen). Add 10 ul for each gel.

*Making SDS Polyacrylamide Gels*
(once the last ingredient, ammonium persulfate, is added, the gel will begin to polymerise)

**Separating (lower) Gel (10% acrylamide)
(change the proportions of water and acrylamide if different from 10% acrylamide)

Lower buffer ---------------------- 1.9 mL
Water------------------------------ 3.1 mL
Acrylamide------------------------ 2.5 mL
TEMED ----------------------------10 uL (add TEMED in the fume hood)
Ammonium Persulfate---------------20 uL
Total ------------------------------7.5 mL

Note that acrylamide in its unpolymerized form is a potent neurotoxin and that gloves must be worn for making gels, setting up the tank for running the gel, and during transferring of the gel

**Stacking Gel (for all percentages of lower gel, use this upper gel)

Upper Buffer -----------------1.25 mL
Water ------------------------3.25 mL
Acrylamide------------------ 0.5 mL
TEMED ----------------------10uL (add TEMED in the fume hood)
Ammonium Persulfate--------- 20uL
Total --------------------------5 mL

*10x Electrophoresis Running Buffer (for the tank)

Tris base---------------------121.4g
Glycine ----------------------567g
SDS --------------------------40g
Water up to ------------------4L

pH 8.3 without adjustment

*4x SDS Loading buffer

Glycerol ---------------------4mL
SDS------------------------ 0.4g
Upper buffer ----------------5mL
up to ----------------------10mL with water

# heat while stirring to dissolve the SDS (don't make the solution boil when heating), and add enough bromophenol blue to make the 1x solution dark enough for easy monitoring when running on a gel
# store at room temperature

*1x Coomasie Blue Dye
(for staining gels that are not used for transferring, or for ensuring that no protein is left after transfer)

Isopropylalcohol ----------25% 500mL
Acetic Acid ---------------10% 200mL
R250 Coomasie Blue--- 0.025% 0.5g
Water ---------------------------1.3L

(destain is 10% Acetic Acid)
# stain 4 hours to ensure all the protein is stained
# destain long enough so that the background is clear (a piece of foam or paper towel may be added to the solution to speed up the destaining)

*10x Towbins Transfer Buffer
(for semi-dry transfer of protein from gel to blot)

250mM Tris -------------------15.1g
1.92M Glycine ----------------72.0g
Water up to --------------------500mL
pH 8.3 without adjustment

*10x Phosphate Buffered Saline—PBS (for removing methanol from blots)

NaCl -----------------------80 g
KCl -------------------------2 g
Na2HPO4 ------------------14.4 g
KH2PO4 -------------------- 2.4 g
water -------------------------800 ml

Adjust pH of PBS Buffer Solution to 7.4 with HCl.
Bring volume to 1 liter, autoclave or sterilize by filtration.
pH of the 1x PBS should be 7.4

*PBS Tween—PBST (for washing blots)

PBS ----------------------------2L
Tween-20 (Brown bottle)-------1mL
stir half an hour

*Amido Black Stain (for viewing all proteins non-specifically after blotting)

Methanol ---------------------40mL
Acetic Acid -------------------10mL
Amido Black 10B ------------0.1g
Up to ------------------------100mL