DNA purification from saliva samples
DNA Extraction
Procedure
Step 1.
Centrifuge whole saliva in a 1.5 mL microcentrifuge tube at 10,000 x g for 5 min.
5 min
@ 10000 𝘨
Step 2.
Discard supernatant.
Step 3.
Resuspend pellet in 1 mL of lysis buffer I.
Step 4.
Add 5 µL proteinase K.
Step 5.
Mix by inversion.
Step 6.
Incubate at 56°C for 2 hours.
2 hr
@ 56 °C
Step 7.
Add 5 µL RNase A and incubate at 37 °C for 15 minutes.
15 min
@ 37 °C
Step 8.
Centrifuge briefly.
Step 9.
Add 100 µL 4M NaCl.
Step 10.
Mixed by inversion for 3 minutes.
3 min
Step 11.
Centrifuge 16,000 × g, 10 minutes.
10 min
@ 16000 𝘨
Step 12.
Carefully transfer the supernatant to a new tube, avoiding the pellet.
Step 13.
Add 0.7 volµme cold isopropanol to the supernatant (e.g. 600 µL supernatant → 420 µL isopropanol
Step 14.
Invert gently several times.
Step 15.
Incubate for 30 minutes at –20 °C.
30 min
@ -20 °C
Step 16.
Centrifuge for 20 minutes at 16,000 × g.
20 min
@ 16000 𝘨
Step 17.
Carefully pour off supernatant.
Step 18.
Add 500 µL 70% ethanol.
Step 19.
Centrifuge for 10 minutes at 16,000 × g.
10 min
@ 16000 𝘨
Step 20.
Carefully pour off ethanol.
Step 21.
Air-dry pellet 30 minutes.
Step 22.
Add 50 µL sterile water.
Step 23.
Incubate at 37 °C for 30 min to fully dissolve.
30 min
@ 37 °C
Required recipes
Lysis buffer I
Recipe calculator ·
Scales ingredients to target volume
Step 1.
Prepare ingredients.
1M Tris (pH 8)
0.5
mL
(10 mM from 1 M stock)
5M NaCl Stock
1
mL
(100 mM from 5 M stock)
0.5M EDTA STOCK
2.5
mL
(25 mM from 0.5 M stock)
10% SDS
2.5
mL
(0.5 % w/v from 10 % w/v stock)
Step 2.
Bring final volume to 50 mL with water.
Used in step 1
1M Tris (pH 8)
Recipe calculator ·
Scales ingredients to target volume
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 water.
Used in step 1
5M NaCl Stock
Recipe calculator ·
Scales ingredients to target volume
Step 1.
Prepare ingredients.
Sodium Chloride
14.61
g
Step 2.
Bring final volume to 50 mL with water.
Used in step 1
0.5M EDTA STOCK
Recipe calculator ·
Scales ingredients to target volume
Step 1.
Prepare ingredients.
EDTA (Ethylenediaminetetraacetic acid)
7.306
g
Step 2.
Bring final volume to 50 mL with water.
Used in step 1
10% SDS
Recipe calculator ·
Scales ingredients to target volume
Step 1.
Prepare ingredients.
Sodium Lauryl Sulfate
5
g
Step 2.
Bring final volume to 50 mL with water.
10% SDS
Recipe calculator ·
Scales ingredients to target volume
Step 1.
Prepare ingredients.
Sodium Lauryl Sulfate
5
g
Step 2.
Bring final volume to 50 mL with water.
1M Tris (pH 8)
Recipe calculator ·
Scales ingredients to target volume
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 water.
5M NaCl Stock
Recipe calculator ·
Scales ingredients to target volume
Step 1.
Prepare ingredients.
Sodium Chloride
14.61
g
Step 2.
Bring final volume to 50 mL with water.
0.5M EDTA STOCK
Recipe calculator ·
Scales ingredients to target volume
Step 1.
Prepare ingredients.
EDTA (Ethylenediaminetetraacetic acid)
7.306
g
Step 2.
Bring final volume to 50 mL with water.