Preparation of backbone vector for cloning 1. Digest vector DNA as follows: - 5 µg DNA (if the restriction enzyme is dilute (<20 U/µl), use only 2 µg DNA) - 5 µl 10x Restriction enzyme buffer - 3 µl restriction enzyme (60 U), Don’t go higher than 3 µl; it may cause star activity - Deionized, sterile water up to 50 µl. Mix carefully, then spin down and incubate at 37 ˚C (or other temperature depending on the enzyme) for at least 3 hours. 2. If the vector is digested with a single enzyme, don’t forget to treat with phosphatase ! Otherwise backbone religation becomes a serious problem. Add 2 µl of Shrimp alkaline phosphatase (SAP, active in any enzyme buffer), spin down and incubate an additional 2 hours at 37 ˚C. 3. The SAP must be heat-inactivated, otherwise it will dephosphorylate the insert as well when ligating. Treat 15 minutes at 65 ˚C to heat-inactivate. 4. Gel purification: Separate the DNA on a preparative 1xTAE (higher yields than TBE gels) agarose gel. I use the 10 well comb and load everything in two wells (25 µl in each). Don’t forget to add loading buffer. Run the gel at 80 V for 1-2 hours (until separation is adequate). 5. Excise the bands and freeze them at –20˚C or process them right away. To isolate the DNA from the agarose gel slice, use the QIAquick gel extraction kit from Qiagen (using spin columns). Elute in the end with 30 µl; ususally 1-2 µl is sufficient for a ligation reaction.