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.