CTAB Precipitation Buffer
CTAB Precipitation Buffer
Purpose:
- Selectively precipitates DNA–CTAB complexes, while many polysaccharides remain soluble.
- Especially important for plants rich in polysaccharides, mucilage, or secondary metabolites.
- Improves purity compared to a direct alcohol precipitation.
Components: Typically 1–2% CTAB in high-salt solution.
- CTAB: detergent that lyses cells and binds polysaccharides.
- Tris-HCl: maintains stable pH.
- EDTA: chelates Mg²⁺, inhibiting DNases.
- NaCl: promotes dissociation of polysaccharides and proteins from DNA.
Preparation
CTAB Precipitation Buffer
Step 1.
Prepare ingredients.
10% CTAB STOCK
0.25
mL
(1 % w/v from 200 % w/v stock)
1M Tris (pH 8)
2.5
mL
(50 mM from 1 M stock)
5M NaCl Stock
1.1688
mL
(40 mM from 1.711157 M stock)
0.5M EDTA STOCK
14.612
mL
(10 mM from 0.034218 M stock)
Step 2.
Bring final volume to 50 mL with distilled water.
Nested recipe inputs
Used in step 1
10% CTAB STOCK
Step 1.
Measure into an appropriately sized container.
CTAB
5
g
(10 % w/v)
Step 2.
Bring final volume to 50 mL with distilled water.
Used in step 1
1M Tris (pH 8)
Step 1.
Weigh out the Tris and add to a media bottle.
Tris Base
12.114
g
Step 2.
Fill media bottle to with ~80% target volume with distilled water.
Step 3.
Add a magnetic stirrer and place on a stirring plate to mix the solution.
Step 4.
Use a pH meter to measure pH. Slowly add concentrated hydrochloric acid (HCl) solution using a Pasteur pipette to reduce the pH to 8.0.
Be careful not to add too much at a time, since the pH will change rapidly.
Step 5.
Once the desired pH has been reached, bring to final volume using distilled water.
Step 6.
Bring final volume to 100 mL with distilled water.
Used in step 1
5M NaCl Stock
Step 1.
Prepare ingredients.
Sodium Chloride
14.61
g
(5 M)
Step 2.
Bring final volume to 50 mL with distilled water.
Used in step 1
0.5M EDTA STOCK
Step 1.
Prepare ingredients.
EDTA
7.306
g
(0.5 M)
Step 2.
Bring final volume to 50 mL with distilled water.