One step hydrolysis of Global DNA for LC-MS/MS detection

1. 1.5 mL sterilized EP tube

2. Sterilized Ultrapure Water

3. Benzonase (Novogen)

4. phosphodiesterase I (B003926-0100 上海生工，Sangon Biotech Co., Ltd)

5. alkaline phosphatase (B600651-0200 上海生工，Sangon Biotech Co., Ltd)

6. Sterilized tips（10 μL，200 μL, 1000 μL）

1. Dry Heater

2. Vortex

Step 1
100 ng/10 uL DNA added 10 μL Global DNA Digestion Solution (Recipes 2)  in 1.5 mL sterilized ep tube. Vortex for at least 10 s.

Step 2

Incubate the mixture at 37 ℃ for 6 h.

• The hydrolysis product could be stored at -20 ℃ for about 3 days.
  
  

Step 3

• For 5mdC detection: 480 μL sterilized ultrapure water was added to the 20 μL of hydrolysis product, for a 50 times dilution before injection.

• For 5hmdC detection: 20 μL hydrolysis product was added to the inject plate for injection directly.  

The injection volume is 2 μL.

Recipe 1. 20 mM Tris-HCl buffer

20 mM Tris-HCl buffer (pH = 7.9)

100 mM NaCl

20 mM MgCl₂

Recipe 2. Global DNA Digestion Solution (5 mL for 500 times）            

250 U benzonase (Novogen)

300 mU phosphodiesterase I (上海生工，Sangon Biotech Co., Ltd)

200 U alkaline phosphatase (上海生工，Sangon Biotech Co., Ltd)

Added in 5 mL 20 mM Tris-HCl buffer

1. E. P. Quinlivan, J. F. Gregory III Anal. Biochem., 2008, 373, 383-385.

2. M. Zhang, L. Quan Anal. Method, 2012, 4, 2515-2521.