Team:HKUST/Protocols/Enzyme digestion

Enzyme digestion (vector, insert)

Purpose: To cut the sequence with specific restriction enzyme

Materials: substrate DNA, restriction enzymes, corresponding 10X buffer, ddH2O, CIP (Calf Intestinal Alkaline Phosphatase, for vector digestion and DNA to be ligated)

Procedure:

a.Add sufficient water to make it a 20 μL reaction.
b.Add 1 μg DNA, 2 μL buffer in total, 0.5 μL each restriction enzyme.
c.Vortex and then spin down.
d.Keep it in 37 °C incubator for one hour. (If needed, incubate for 1.5-2hours)
e.If the substrate DNA is vector or pieces to be ligated, add 0.5 ul (for 20ul reaction) CIP after incubated for 1.5-2 hours, and then incubate at 37 °C for another 30mins.
f.Check the result by gel electrophoresis.

Tips:

A. Restriction enzymes are easy to be denatured, so do not leave it at room temperature.
B. If two restriction enzymes must be added with different buffer, digest the DNA with respective restriction enzyme sequentially. Incubate for one hour after each enzyme added.

• Agarose gel preparation and gel electrophoresis
• PCR (Taq, Vent)
• PCR product clean-up
• Enzyme digestion (vector, insert)
• Plasmid DNA extraction
• Gel cutting
• Gel extraction
• Ethanol precipitation
• Ligation
• Transformation to E.coli
• Transformation to yeast
• Cell lysis
• Yeast genomic DNA extraction
• Western blotting
• GFP testing