22 Expression in Eukaryotic Cells: Gene Transfer Using Retroviral Vectors
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Subprotocol 5
Assay for the Presence of the DNA Insert
The presence of the insert can easily be confirmed by digestion of the plasmid DNA recovered from the miniprep. Plasmids are digested with the same
restriction enzymes used prior to the ligation of the insert into the vector. In
the following, a standard protocol is given which assumes that the EcoRI
and BamHI sites in the multiple cloning region of pLXSN have been used.
Materials
• Ethanol precipitated plasmid DNA
• EcoRI and BamHI (10 U/J.tl)
• Digestion buffer
• Ultra pure water
• TE
• RNase stock solution (1 mg/ml)
Procedure
1. Resuspend the ethanol precipitated plasmid DNA in 60 Jll TE.
2. Transfer 14 Jll to a new microcentrifuge tube.
3. Add 2 Jll EcoRI, 2 Jll BamHI, 3 Jll digestion buffer and 9 Jll ultra pure
water.
4. Incubate 2 hours at 37°C.
5. Add 3 Jll RNase and incubate for an additional 15 minutes at 37°C.
6. Analyze the samples on a horizontal agarase gel and detect the plasmid
DNA and the insert DNA with ethidium bromide.