Sample Preparation and composition

ACK Lysis Buffer Preparation

ACK Lysis Buffer is used to lyse red blood cells. To prepare 1L of ACK Lysis Buffer the following components are required;

Component	Amount	Concentration
NH4Cl (mw: 53.49 g/mol)	8.02 g	0.15 M
KHCO3 (mw: 100.12 g/mol)	1 g	0.01 M
Na2EDTA (mw: 372.24 g/mol)	37 mg	0.0001 M

ACK Lysis Buffer Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 8.02 g of NH4Cl to the solution.
3. Add 1 g of KHCO3 to the solution.
4. Add 37 mg of Na2EDTA to the solution.
5. Adjust the pH to 7.2-7.4.
6. Add distilled water until volume is 1 L.
7. Store for up to 6 mo at room temperature.

Alkaline Sodium Thiosulfate Preparation

To prepare 1L of Alkaline Sodium Thiosulfate the following components are required;

Component	Amount	Concentration
Sodium thiosulfate (mw: 158.11 g/mol)	200 g	1.265 M
NaOH (mw: 40 g/mol)	4 g	0.1 M

Alkaline Sodium Thiosulfate Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 200 g of Sodium thiosulfate to the solution.
3. Add 4 g of NaOH to the solution.
4. Add distilled water until volume is 1 L.

BAC DNA Microinjection Buffer Preparation 

BAC injection buffer functions to stabilize BAC DNA in preparation for microinjection. To prepare 1L of BAC DNA Microinjection Buffer the following components are required;

Component	Amount	Concentration
Tris (pH 7.5) (mw: 121.14 g/mol)	1.211 g	0.01 M
EDTA (mw: 292.24 g/mol)	29 mg	0.0001 M
NaCl (mw: 58.44 g/mol)	5.844 g	0.1 M

BAC DNA Microinjection Buffer Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 1.211 g of Tris (pH 7.5) to the solution.
3. Add 29 mg of EDTA to the solution.
4. Add 5.844 g of NaCl to the solution.
5. Adjust the pH to 7.4.
6. Add distilled water until volume is 1 L.
7. Filter the injection buffer (through a 0.2-U filter) and store at 4?C.

DEAE-dextran Preparation

To prepare 1L of DEAE-dextran the following components are required;

Component	Amount	Concentration
DEAE-dextran (mw: 500000 g/mol)	50 g	0.0001 M

DEAE-dextran Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 50 g of DEAE-dextran to the solution.
3. Add distilled water until volume is 1 L.
4. Sterilize the solution by autoclaving for 20 minutes at 15 psi (1.05 kg/cm2) on liquid cycle. Autoclaving also assists dissolution of the polymer. The molecular weight of the DEAE-dextran originally used for transfection was >2 x 106. Although this material is no longer available commercially, it is still occasionally found in chemical storerooms. The older batches of higher-molecular-weight DEAE-dextran are more efficient for transfection than the lower-molecular-weight polymers currently available.

EDTA Solution Preparation

Metal Stripping Solution is comprised of EDTA, which can act as a metal chelating agent. To prepare 1L of EDTA Solution the following components are required;

Component	Amount	Concentration
EDTA (mw: 292.24 g/mol)	11.7 g	0.04 M

EDTA Solution Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 11.7 g of EDTA to the solution.
3. Add distilled water until volume is 1 L.
4. Slowly adjust the pH to 8.0 with 2 M NaOH. (Unless using the salt, EDTA often requires extended time and basic pH conditions to completely dissolve.) Store for up to 3 mo at 4°C.

Elution Buffer Preparation

Elution buffer is commonly used in many applications such as affinity chromatography, immunoprecipitation, protein purification, and DNA extraction to elute proteins or DNA from a ligand or membrane. To prepare 1L of Elution Buffer the following components are required;

Component	Amount	Concentration
NaCl (mw: 58.44 g/mol)	23.38 g	0.4 M

Elution Buffer Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 23.38 g of NaCl to the solution.
3. Add distilled water until volume is 1 L.
4. Filter the solution through a nitrocellulose filter (0.45-?m pore size) and store at room temperature.

Gey’s Balanced Salt Solution Preparation

Gey’s Balanced Salt Solution can be used for cell suspension or as washing solution. To prepare 1L of Gey’s Balanced Salt Solution the following components are required;

Component	Amount	Concentration
CaCl2·2H2O (mw: 147.01 g/mol)	220 mg	0.0015 M
KCl (mw: 74.55 g/mol)	370 mg	0.005 M
KH2PO4 (mw: 136.09 g/mol)	30 mg	0.0002 M
MgCl2·6H2O (mw: 203.3 g/mol)	210 mg	0.001 M
MgSO4·7H2O (mw: 246.47 g/mol)	70 mg	0.0003 M
NaCl (mw: 58.44 g/mol)	8 g	0.1368 M
NaHCO3 (mw: 84.01 g/mol)	227 mg	0.0027 M
Na2HPO4 (mw: 141.96 g/mol)	120 mg	0.0008 M
D-glucose (mw: 180.16 g/mol)	1 g	0.0056 M

Gey’s Balanced Salt Solution Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 220 mg of CaCl2·2H2O to the solution.
3. Add 370 mg of KCl to the solution.
4. Add 30 mg of KH2PO4 to the solution.
5. Add 210 mg of MgCl2·6H2O to the solution.
6. Add 70 mg of MgSO4·7H2O to the solution.
7. Add 8 g of NaCl to the solution.
8. Add 227 mg of NaHCO3 to the solution.
9. Add 120 mg of Na2HPO4 to the solution.
10. Add 1 g of D-glucose to the solution.
11. Add distilled water until volume is 1 L.
12. Sterile filter and store in 500-mL aliquots at 4?C.

H-50 Buffer Preparation

H-50 buffer is commonly used for washing and resuspending cells. It contains HEPES, one of the Good’s zwitterionic buffers. To prepare 1L of H-50 Buffer the following components are required;

Component	Amount	Concentration
HEPES (mw: 238.3 g/mol)	4.76 g	0.02 M
KCl (mw: 74.55 g/mol)	3.73 g	0.05 M
MgSO4 (mw: 120.37 g/mol)	120 mg	0.001 M
NaCl (mw: 58.44 g/mol)	580 mg	0.01 M
NaHCO3 (mw: 84.01 g/mol)	420 mg	0.005 M
NaH2PO4 • 2H2O (mw: 156.01 g/mol)	156 mg	0.001 M

H-50 Buffer Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 4.76 g of HEPES to the solution.
3. Add 3.73 g of KCl to the solution.
4. Add 120 mg of MgSO4 to the solution.
5. Add 580 mg of NaCl to the solution.
6. Add 420 mg of NaHCO3 to the solution.
7. Add 156 mg of NaH2PO4 • 2H2O to the solution.
8. Add distilled water until volume is 1 L.
9. Adjust the pH to 7.0 with HCl or NaOH, as appropriate. Autoclave and store cold or frozen.

Lysis Solution Preparation

 To prepare 1L of Lysis Solution the following components are required;

Component	Amount	Concentration
NaCl (mw: 58.44 g/mol)	146.1 g	2.5 M
Na2EDTA (mw: 372.24 g/mol)	37.2 g	0.1 M
Tris base (mw: 121.14 g/mol)	1.2 g	0.01 M
NaOH (mw: 40 g/mol)	8 g	0.2 M
N-lauroylsarcosine sodium salt (mw: 293.38 g/mol)	10 g	0.0341 M

Lysis Solution Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 146.1 g of NaCl to the solution.
3. Add 37.2 g of Na2EDTA to the solution.
4. Add 1.2 g of Tris base to the solution.
5. Add 8 g of NaOH to the solution.
6. Add 10 g of N-lauroylsarcosine sodium salt to the solution.
7. Adjust pH to 10.
8. Add distilled water until volume is 1 L.
9. Store at room temperature.

Osmium Tetroxide Solution Preparation

Osmium tetroxide solution is used as a fixative in electron microscopy.  To prepare 1L of Osmium Tetroxide Solution the following components are required;

Component	Amount	Concentration
osmium tetroxide (mw: 254.23 g/mol)	40 g	0.1573 M

Osmium Tetroxide Solution Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 40 g of osmium tetroxide to the solution.
3. Add distilled water until volume is 1 L.
4. Make sure that the lid is tight and well sealed. Wrap in aluminum foil and stand the bottle in a larger container, sealed with a screw cap. Leave the osmium tetroxide to dissolve for 2-3 days at 4°C. Dilute 1 in 5 in double-distilled H2O before use. Osmium tetroxide solutions may be stored at -20°C for up to 6 months.

Paraformaldehyde Solution (8%) Preparation

Paraformaldehyde solution is commonly used to fix cells or tissues. To prepare 1L of Paraformaldehyde Solution (8%) the following components are required;

Component	Amount	Concentration
paraformaldehyde (mw: 30.03 g/mol)	80 g	2.664 M

Paraformaldehyde Solution (8%) Preparation steps 

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 80 g of paraformaldehyde to the solution.
3. Heat to 60°C and add 2-3 drops of 1 N NaOH.
4. Add distilled water until volume is 1 L.
5. Store this stock solution at 4°C.

TE Buffer 10X Preparation

TE buffer is used as a protective measure against DNA and RNA degredation, storing the two molecules and maintaining proper pH levels. To prepare 1L of TE Buffer 10X the following components are required;

Component	Amount	Concentration
Tris-Cl (desired pH) (mw: 157.594 g/mol)	15.759 g	0.1 M
EDTA (pH 8) (mw: 292.24 g/mol)	2.92 g	0.01 M

TE Buffer 10X Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 15.759 g of Tris-Cl (desired pH) to the solution.
3. Add 2.92 g of EDTA (pH 8) to the solution.
4. Add distilled water until volume is 1 L.

Trichloroacetic Acid (TCA) Preparation 

To prepare 1L of Trichloroacetic Acid (TCA) the following components are required;

Component	Amount	Concentration
Trichloroacetic acid (TCA) (mw: 163.39 g/mol)	2202.64 g	13.481 M

Trichloroacetic Acid (TCA) Preparation steps

1. Prepare 800 mL of distilled water in a suitable container.
2. Add 2202.64 g of Trichloroacetic acid (TCA) to the solution.
3. Add distilled water until volume is 1 L.
4. The resulting solution will contain 100% (w/v) TCA.