2 Materials
All solutions and media should be sterilized by autoclaving and
prepared using water purified to 18.2 MΩ/cm.2.1 Buffers and
Solutions
- Tris Acetate Ethylenediaminetetraacetic acid (EDTA) buffer
(TAE) stock solution (50): dissolve 242 g Tris-base and
18.5 g EDTA disodium salt in 500 mL H 2 O while stirring;
add 57.1 mL glacial acetic acid and bring the volume to 1 L
with H 2 O. - 10DNA loading dye: 30% (vol/vol) glycerol in TAE buffer
1 . Add 0.3 mg/mL Xylenol Orange dye, mix well, and
sterilize by filtration. - 0.1 M sodium phosphate buffer, pH 7.0: mix 57.7 mL of
Na 2 HPO 4 and 42.3 mL of NaH 2 PO 4 and bring the volume
to 1 L with H 2 O. - Elastin-Congo Red suspension: 2 g/mL of Elastin-Congo red
dissolved in 10 mM sodium phosphate buffer, pH 7.0 - M9 buffer: 15.1 g/L Na 2 HPO 4 , 3 g/L KH 2 PO 4 , 5 g/L NaCl,
0.25 g/L MgSO 4 ·7H 2 O. - Phosphate-buffered saline (PBS): dissolve 8 g of NaCl, 0.2 g of
KCl, 1.44 g of Na 2 HPO 4 , and 0.24 g of KH 2 PO 4 in 800 mL of
H 2 O. Adjust pH to 7.4 with HCl. Add H 2 O to 1 L. Sterilize by
autoclaving. - MPBS: PBS containing 2% (wt/vol) skim milk powder.
- ELISA stop solution: 1 M H 2 SO 4.
- Fractionation buffer: 200 mM Tris–HCl, 20% (wt/vol)
sucrose, 1 mM EDTA, and 0.05 mg/mL lysozyme. - Ampicillin (Ap) stock solution: 50 mg/mL Ap dissolved in
H 2 O. Sterilize by filtration and store at 20 C. - Kanamycin (Km) stock solution: 25 mg/mL Km dissolved in
H 2 O. Sterilize by filtration and store at 20 C. - Glucose stock solution: 25% (wt/vol) glucose dissolved in
H 2 O. Sterilize by filtration and store atþ 4 C. - PEG-NaCl solution: 20% (wt/vol) PEG 6000, 2.5 M NaCl.
- Potassium phosphate stock solution for Terrific Broth (TB)
medium: 23.1 g/L KH 2 PO 4 , 125.4 g/L K 2 HPO 4. Sterilize
by filtration. - Cholesterol stock solution: 5 mg/mL cholesterol dissolved in
ethanol. Sterilize by filtration. - Nematode bleaching solution: add 200μL of bleach and 25μL
of 4 M NaOH or KOH to 775μLofH 2 O.
328 Soumya Palliyil