2 X CTAB
| /100mL | /500mL | /1L | ||
|---|---|---|---|---|
| ddH2O | (mL) | 86 | 430 | 860 |
| CTAB | (g) | 2 | 10 | 20 |
| Polyvinylpyrrolidone | (g) | 1 | 5 | 10 |
| 1M Tris-HCl pH8.0 | (mL) | 10 | 50 | 100 |
| 0.5M EDTA 8.0 | (mL) | 4 | 20 | 40 |
| NaCl (FW 58.44g) | (g) | 8.2 | 40.9 | 81.8 |
- Slowly add components in order and allow each to dissolve.
- Use mild heat to help facilitate dissolving – may take a couple hours.
- Autoclave.
- (CTAB – Cetyl-trimethyl-ammonium-bromide)
10% CTAB
| /100mL | /500mL | ||
|---|---|---|---|
| ddH2O | (mL) | 100 | 500 |
| CTAB | (g) | 10 | 50 |
| NaCl | (g) | 8.18 | 40.9 |
- Dissolve CTAB before the salt.
- Use mild heat to help dissolve – may take a couple hours.
- Autoclave.
0.5M EDTA pH 8.0
- Add 186.1 g of Disodium-ethylenediamine-tetra-acetate to 800mL H2O.
- pH to 8.0, using NaOH pellets, and adjust final volume to 1 L.
- Autoclave
1M Tris-HCl pH 8.0
- Add 121.1 g of Tris base in 800mL H2O.
- pH to 8.0, using HCl, and adjust final volume to 1 L.
- Autoclave.
First make and autoclave these component solutions:
- 1M Tris Base
- 3M KCl
- 0.5M EDTA (pH 8.0)
- DI Water
- 5M NaCl
10x HB (Homogenization buffer):
- Use about 30 mL/extraction
- Store in refrigerator
Final solution should be
- 0.1 M Tris base
- 0.8 M KCl
- 0.1 M EDTA
- 10 mM spermidine
- 10 mM spermine
- adjust pH to 9.4-9.5 with NaOH
To prepare 500 mL
- 50 mL of 1 M Tris (pH 9.5)
- 133 mL of 3 M KCl
- 100 mL of 0.5 M EDTA
- 780 µL of spermidine
- 1.741 g of spermine
Wash Buffer (AKA 1X HB):
Make fresh on day of use
- 1/10th volume 10x HB
- 0.5 M Sucrose
Lysis Buffer:
Use 3 mL per tube (step 12)
- 10 mM Tris-HCl
- 400 mM NaCl
- 2 mM EDTA (pH 8.0)
Proteinase K Solution:
Make fresh on day of use
-
Use 0.5 mL per tube (step 13)*
- 1 mg/mL Proteinase K
- 1% SDS
- 2mM EDTA (pH 8.0)