Preparation of insert DNA fragments

Restriction endonuclease (0.5-2 units) in appropriate amounts, and incubate under optimum conditions to obtain complete digestion (manufacturers recommendations) in a final volume of 20 /zl. 

Heat 70°C for 10 min. to inactivate enzyme (except Taql and other thermostable enzymes). 

If the enzyme gives 3 - or 5 -sticky ends which cannot be ligated directly into a site in M13mp7 (e.g. Hhal, Hinfl), the fragment termini can be converted into blunt ends  

linker 5 -GGAATTCC as purchased, terminates in 5 -CCTT AAGG-5  

OH groups. For ligation to restriction fragments 5 -phosphoryIated ends are required and the first step is the phosphorylation of these groups using polynucleotide kinase  

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