Fill in Reactions (Klenow and T4 DNA polymerase)

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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.