Assessing the Quality of Total RNA by Gel Electrophoresis

1. Agarose (Invitrogen; cat. no. 15510-027) Store in a cool and dry place at RT; stable for minimum 5 yr.

2. Heating block suitable for 0.5-mL tubes.

3. Gel loading tank.

4. Power pack.

5. Ultraviolet light box.

6. Photographic equipment.

7. Denaturing loading buffer (see Subheadings 2.4.2.1. and 2.4.2.2.).

8. DNA ladder/marker 200 base pairs (bp) to 3 kbp: ^X174 DNA/HaeIII (Promega; cat. no. G1761; store at -20°C; stable for 1 yr) and 1-kb DNA ladder (Promega; cat. no. G5711; store at -20°C, stable for 1 yr).

9. Ethidium Bromide tablets (Sigma-Aldrich; cat. no. E-2515) Store at RT; stable for minimum 5 yr. Safety Information: harmful if swallowed; very toxic by inhalation. Irritating to eyes, to respiratory system, and to skin. Keep container tightly closed and in a well-ventilated place.

10. 10X TBE stock solution (see Subheadings 2.4.2.3. and 2.4.2.4.). Alternatevely (Invitrogen; cat. no. 15581-028). Store at RT. Safety information: toxic. Harmful by inhalation and in contact with skin. Irritating to eyes and to skin.

2.4.2.1. Denaturing Loading Buffer

1. 10X MOPS (3[N-Morpholino]propanesulfonic acid; Sigma-Aldrich; cat. no. M-8899) Store at RT; stable for 3 yr. Safety Information: Irritating to eyes, to respiratory system, and to skin.

2. N,N-Dimethylformamide (Sigma-Aldrich; cat. no. D-4551) Store at RT; stable for minimum 5 yr. Safety Information: combustible. Teratogen, target organs: liver and kidneys. May cause harm to the unborn child. Harmful by inhalation and in contact with skin. Irritating to eyes and to skin.

3. Mixed-Bed Resin (Sigma-Aldrich; cat. no. M-8032) Store at RT, stable for minimum 3 yr.

4. Formaldehyde 37% (BDH; cat. no. 43753) Store at RT, stable for minimum 5 yr. Safety Information: toxic by inhalation, in contact with skin, and if swallowed. Causes burns. Danger of very serious irreversible effects. May cause sensitiza-tion by skin contact.

5. Glycerol (BDH; cat. no. 10118) Store at RT, stable for minimum 5 yr.

6. Bromophenol Blue (Sigma-Aldrich; cat. no. B-8026) Store at RT, stable for minimum 5 yr. Safety Information: avoid contact and inhalation.

2.4.2.2. Method for Preparing Denaturing Loading Buffer

1. Prepare 10X loading buffer (50 % glycerol, 0.1 mg/mL bromophenol blue sodium salt) as follows:

2. Add 0.2 mg of bromophenol blue sodium salt to 500 ||L of DEPC H2O and mix.

3. Add 500 |L of glycerol and mix thoroughly.

5. Prepare deionized formamide as follows:

6. Add 1 g of mixed bed resin to 50 mL of formamide.

8. Filter through filter paper.

10. Prepare in bulk 10 |L of loading buffer for each 1-1.5 |g/|L sample of total RNA, cRNA, or fragmented cRNA as follows:

Deionized formamide 5.00 | L

Formaldehyde 37% 1.75 |L 10X loading buffer (50% glycerol, 0.1 mg/mL Bromophenol Blue) 1.00 |L

Final volume 10.0 |L

11. Freeze in 100- to 200-|L aliquots and keep at -20°C.

12. Make a 10mg/mL ethidium bromide solution when ready to add buffer to samples.

13. Add 0.5 |L of ethidium bromide (10 mg/mL)/100 |L defrosted denaturing loading buffer just before adding the buffer to the samples.

2.4.2.3. 10X TBE Stock Solution

2. TRIS base (Invitrogen; cat. no. 15504-020). Store at RT; stable for minimum of 2 yr. Safety Information: toxic by inhalation, in contact with skin, and if swallowed. Irritating to eyes, respiratory system.

3. Boric acid (BD; cat. no. 100583R). Store at 15-30°C; stable for minimum of 2 yr. Safety Information: irritating by inhalation, in contact with skin, and if swallowed.

4. Ethylenediamine tetra-acetic acid (EDTA) di-Sodium Salt, pH 8.0 (Sigma-Aldrich; cat. no., E-7889). Store at RT, stable for 1 yr.

2.4.2.4. Method for Preparing 10X TBE Stock Solution

1. Make a solution of: (1) 108 g TRIS, (2) 55 g boric acid, (3) 40 mL 0.5 M EDTA, and (4) DEPC H2O to 1000 mL.

2. Filter through a 0.2-|im vacuum filter unit. Store at 4°C and shield from light.

Was this article helpful?

0 0

Post a comment