Fill in Reactions (Klenow and T4 DNA polymerase)
Klenow Fill in (for 5’ Protruding Ends)
10 X Nick Translation buffer: (200 uL)
100 uL 1 M Tris (pH 7.2) (0.5 M Tris, pH 7.2)
20 uL 1 M MgSO4 (0.1 M MgSO4)
0.4 uL 0.5 M DTT (1 mM DTT)
100 ug BSA (500 ug / mL BSA)
1. Mix up the reaction:
5 uL DNA (0.5 – 2 ug total)
2 uL 10 X Nick Translation Buffer
1 uL 2 mM dATP
1 uL 2 mM dCTP
1 uL 2 mM dTTP
1 uL 2 mM dGTP
1 uL Klenow (5 U)
2 uL BSA (500 ug / mL)
6 uL DI H2O
2. Incubate at Room Temp. for 1 hour.
3. Phenol / Chloroform extraction.
4. Chloroform Extraction.
5. Adjust salt, add tRNA.
6. Ethanol Precipitation.
T4 Polymerase Fill in (for 3’ Protruding Ends)
10 X T4 Polymerase Buffer (200 uL)
10 uL 1 M DTT
140 uL 1 M Tris (pH 7.4)
20 uL 1 M MgCl2
30 uL DI H2O
1. Mix up the reaction:
9 uL DNA (0.5 – 2 ug total)
2 uL 10 X T4 Polymerase Buffer
3 uL DI H2O
1 uL 2 mM dATP
1 uL 2 mM dCTP
1 uL 2 mM dTTP
1 uL 2 mM dGTP
2 uL T4 DNA Polymerase (1 U / uL) (use 2 – 5 U)
2. Incubate reaction for 15 minutes at 37 C.
3. Incubate reaction for 10 minutes at 70C.
4. Phenol / Chloroform Extraction.
5. Chloroform Extraction.
6. Adjust salt, add tRNA.
7. Ethanol Precipitation.