CTAB Lysis Buffer
CTAB is a detergent that disrupts cell membranes and forms complexes with polysaccharides, helping to separate them from DNA.
Tris-HCl buffers the pH to stabilize DNA.
EDTA chelates divalent cations (like Mg²⁺), which inactivates DNases.
NaCl aids in removing polysaccharides and proteins.
β-mercaptoethanol reduces disulfide bonds, preventing oxidation of phenolic compounds.
PVP (polyvinylpyrrolidone) binds polyphenols that might otherwise co-precipitate with DNA.
Preparation
CTAB Lysis Buffer
Step 1.
Measure into an appropriately sized container.
10% CTAB STOCK
5
mL
1M Tris (pH 8)
5
mL
(100 mM from 1 M stock)
0.5M EDTA STOCK
29.224
mL
(20 mM from 0.034218 M stock)
5M NaCl Stock
29.22
mL
(1 M from 1.711157 M stock)
2-Mercaptoethanol
100
μL
Polyviny|pyrrolidone
0.5
g
(1 % w/v)
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
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.
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.