PCR Purification

Polymerase Chain Reaction (PCR)

Procedure

Step 1. Bind: Add 5 volµmes of binding buffer to 1 volµme of sample. Count the loading buffer in the sample volµme: 30 µL of sample takes 150 µL of buffer.
Step 2. Load: Put the sample with binding buffer in a spin colµmn and spin 30–60 s. Discard the flow-through.
Step 3. Wash: Add 700 µL of wash buffer, spin 1 min, and discard the flow-through. If a blue tint stays on the silica, wash a second time.
Step 4. Dry spin: Spin the empty colµmn 1–2 min. Don’t skip this; leftover ethanol hurts the sequencing reaction.
Step 5. Elute: Move the colµmn to a clean tube. Add 20–30 µL of elution buffer or water, warmed to ~50 °C, to the center of the membrane. Let it stand 2 min, then spin 1 min.

Required recipes

Wash Buffer

Recipe calculator · Scales ingredients to target volume
Step 1. Add to appropriate container.
Ethanol, 200 proof
80 mL
5M NaCl Stock
400 μL
Tris, 1M, pH 7.5
200 μL
Step 2. Bring final volume to 100 mL with distilled water.
Used in step 1

5M NaCl Stock

Recipe calculator · Scales ingredients to target volume
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

Tris, 1M, pH 7.5

Recipe calculator · Prepares solution at target volume
Step 1. Add approximately 35 mL distilled water.
Step 2. Dissolve.
Tris Base
12.114 g
Step 3. Adjust to pH 7.5 with concentrated HCl (~12 M). Expect to need roughly 6–7 mL.
Step 4. Bring final volume to 50 mL with distilled water.
Step 5. Autoclave at 121 °C for 20 min (liquid cycle).

Binding Buffer

Recipe calculator · Scales ingredients to target volume
Step 1. Add approximately 35 mL distilled water.
Step 2. Add to water
Guanidine Hydrochloride
47.8 g
Sodium Acetate, 3M, pH 5.2
1 mL
Step 3. Dissolve Gu-HCL.
Step 4. Add to container
Isopropanol
30 mL
Step 5. Bring final volume to 100 mL with distilled water.
Used in step 2

Sodium Acetate, 3M, pH 5.2

Recipe calculator · Scales ingredients to target volume
Step 1. Weigh into appropriate container.
Sodium Acetate, Trihydrate
40.8 g
Step 2. Add approximately 60 mL distilled water.
Step 3. Adjust to pH 5.2 with glacial acetic acid. Expect to need roughly 6–8 mL
Step 4. Bring final volume to 100 mL with distilled water.
Step 5. Filter-sterilize through a 0.22 µm filter.