1. Prepare bulk digestion mix using the appropriate enzyme (PstI, SalI, etc.) and correct enzyme buffer.
STOCK [FINAL] Per 30 μl RXN
ddH2O ó 1.75 μl
10X Buffer 1X 3.00 μl
0.1 M Spermidine 2.5 mM 0.75 μl
Enzyme (10 U/μl) 25 U 2.50 μl
Plasmid (1 μg/μl) 22 μg 22.00 μl
2. Add bulk mix to 500 μl microfuge tube containing plasmid and incubate at 37•C for 2-3 hours. A 37•C oven
works best because there is minimal condensation on the sides of the tube.
3. Stop reaction by adding 6 μl of 5X SGB which contains only the xylene cyanole dye.
4. Remove 1 μl (650 ng of plasmid) to use for determining MW of insert. Electrophorese in 1% standard agarose
gel with HaeIII digest of ΦX174 as MW standards (see p. 23).
5. Prepare a 1.1% LMP agarose gel. Heat the agarose a little slower than regular agarose to minimize foaming.
Once the gel has set, place at 4•C to cool. The gel, running buffer, stain and destaining solutions should be kept
at 4•C prior to and during the run. Include EtBr in the gel and running buffer.
6. Remove the gel from the refrigerator and load the samples (can be done at RT). Place into pre-cooled gel
apparatus and run in the cold at 40 mA until the dye has migrated about 2 cm (on a 1.1% gel, pUC18, 2700 bp,
will run just below the xylene cyanole dye). Check separation with portable UV lamp after 30 min (if running
in a minigel).
7. When visualizing the bands, it is best to minimize exposure to UV by either using a hand-held long wave UV
lamp or by leaving the gel on a UV transparent tray and placing on a transilluminator.
8. Quickly mark the insert bands by pushing a 1.5 inch section of a plastic soda straw into the gel around each
9. Once all the inserts have been marked, turn off the UV light. Remove each straw from the gel and force the
agarose plug into a screw cap tube using a P-200 pipetteman (place the barrel into the end of the straw and
depress the plunger to force the plug out of the straw into the tube). Sarstedt tubes (# 72.694/006) are good
since they seal tightly and have a good writing surface.
10. Assuming you know the MW of the insert and had 100% digestion, dilute each sample in dH2O to the desired
concentration (10 ng/μl). We only approximate the final volume using the markings on the Sarstedt tubes.
11. Mix the agarose-water mixture by heating at 65-70•C for 5-10 min. Vortex and store at 4•C in tightly sealed
tubes. Under these conditions, inserts are stable for oligolabeling for several years.