DNA end blunting

Reagents

DNA end blunting is a very common approach in molecular cloning. Although the advances in PCR technology make it easy to engineer any restriction enzymatic sites, straight cloning is still the first choice whenever possible. Adding restriction sites by PCR usually requires an extra sequencing step to make sure no mutations are introduced. In straight cloning, some time only one restriction site is compatible. Then one end sticky, one end blunt approach can be tried. Under some extreme conditions, double blunt cloning is required with both ends are blunt.

For one end sticky, one end blunt approach:

1. For example, EcoR I can be used as sticky end and Sal I can be used for blunting.

2. Digest 1 ug DNA with Sal I to complete.

3. Clean the DNA and elute DNA with 30 ul TE buffer.

4. Add 3 ul 10 X polymerase buffer, 1 ul T4 DNA polymerase, 0.6 ul BSA (Promega), 0.6 ul dNTP (10 mM each) and mix.

5. Incubate at 37C for 1 hr.

6. Clean the DNA and elute DNA with 30 ul TE buffer.

7. Digest with EcoR I to complete.

8. Either clean the DNA for ligation or extract the right band for ligation.


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Posted by ebiomethods Mon, 22 Dec 2008 22:08:29
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