Methods in Molecular Biology • 16 Enzymes of Molecular Biology

24 Maunders

3.3.1.2. EQUIPMENT REQUIRED

1. 1.5-mL Microfuge tubes.


2. Microfuge.


3. Boiling water bath or dry heating block.


4. Water bath (37°C).


5. Pipets and sterile pipet tips.


6. Ice bucket.

3.3.1.3. PROTOCOL

1. Dilute 50 ng probe DNA to 34 ~tL with sterile water.


2. Denature by boiling for 3 rain. Spin down condensation and stand on ice.


3. Add l0 ~L of reaction buffer.


4. Add 5 laL (50 [xCi) a-32P dATP.


5. Add 1 laL (2 U) Klenow fragment.


6. Incubate at 37°C for 30 min or at room temperature for 1-2 h.


7. Add 200 ]aL sterile water, and terminate the reaction by boiling for 3 min.


8. Stand the reaction on ice and use (unpurified) within 10 min.
   3.3.2. Protocol for In-Filling Cohesive Termini

3.3.2.1. MATERIALS REQUIRED

1. Target DNA.


2. Reaction buffer:

• 70 mM Tris-HC1, pH 7.5


• 70 mMMgC12


• 10 mMDithiothreitol


• 250 laM dATP


• 250 ~ dGTP


• 250 laM dCTP


• 250 gM dTTP
  N.B. Alternatively, the in-filling reaction can be performed in most
  restriction endonuclease buffers.

3. 500 mM NaC1.


4. Klenow fragment (diluted to 0.5 U/IaL in storage buffer).


5. Sterile distilled water.

3.3.2.2. EQUIPMENT REQUIRED

1. 1.5-mL Microfuge tubes.


2. Water bath (25°C).


3. Water bath or dry heating block (70°C).


4. Pipets and sterile pipet tips.