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BQ-042-101-05
Revision : 2 (2016-07-04)
XII.
Protocols
Before you begin
1.
Completely dissolve Proteinase K (KB-0111) in 1,250 ul of nuclease-free water. Dissolved Proteinase
K should be stored at 4℃.
2.
Completely dissolve RNase A (KB-3101) in 600 ul of nuclease-free water. Dissolved RNase A should
be stored at 4℃.
3.
Buffer ② (Binding) contains chaotropic salt. You should take the appropriate laboratory safety
precautions and wear gloves when handling.
4.
Add the correct amount of absolute ethanol to Buffer ③ (1
st
Washing) and Buffer ④ (2
nd
Washing).
a. DNA Extraction from Whole Blood for Mini/Midi/Maxi scale
1.
Add 20 μl (mini)/ 100 μl (midi)/ 200 μl (maxi) of proteinase K (see “Before you begin”) to each specific
tube format.
a. (Mini) Add proteinase K to a 1.5 ml or 2 ml tube.
b. (Midi) Add proteinase K to a 15 ml tube.
c. (Maxi) Add proteinase K to a 50 ml tube.
2.
Apply 200 μl (mini)/ 2 ml (midi)/ 4 ml (maxi) of whole blood and buffy coat to the tube containing
proteinase K.
(Note) If the sample volume is less than indicated volume above, make the total volume 200 μl (mini)/
2 ml (midi)/ 4 ml (maxi) by adding 1X PBS to achieve maximum lysis efficiency and yield.
3.
(Lysis: 3-4)
Add 200 μl (mini)/ 2 ml (midi)/ 4 ml (maxi) of Buffer ② (Binding) to each sample and mix
immediately and thoroughly using a vortex mixer. You must completely resuspend the sample to
achieve maximum lysis efficiency.
4.
Incubate at 60℃ for 10 min.
5.
(DNA Precipitation)
Add 400 μl (mini)/ 4 ml (midi)/ 8 ml (maxi) of absolute ethanol and mix well using a
