2 Enzymatic Modification of DNA and RNA
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Materials
• water bath 75°C
• Alternatively: Thermal cycler, programmed to 22°C/30 min, 75°C/10
min, maximum temperature slope
• DNA (0.1-5 ~g in 20 ~1) which has been digested with restriction enzymes
that create recessed 3' ends. Klenow enzyme works well in virtually all
restriction enzyme buffers. Therefore, the filling in reaction can be performed after DNA digestion in the same reaction mix.
• 10x buffer (if necessary) is 0.5 M Tris-HCl pH7.6, 0.1 M MgCh
• dNTP solution: 1 mM dATP, 1 mM dCTP, 1 mM dGTP, 1 mM dTTP in
water. This solution should be stored insmall aliquots at -20°C.
• Klenow enzyme (various suppliers; usually 1 U/~1)
• TE buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA)
• Chaotropic buffer, silica-gel spin columns
Procedure
Filling in recessed 3' ends with Klenow enzyme
Supplies
Reagents
and solutions
1. After completion of restriction enzyme digest, add the following to the Klenow enzyme
reaction:
DNA (RE digest)
dNTP
Klenow enzyme
2. Incubate at room temperature for 30 min.
0.1-5 ).lg
1 nmol each
1U
3. Inactivate the enzyme by heating to 75°C for 10 min.
4. Increase volume to 100 ~1 with TE.
5. Purify the DNA by using a chaotropic buffer/silica-gel spin column system according to the manufacturer's instructions.