Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA · Toll Free in USA 800-356-9526 · 608-274-4330 · Fax 608-277-2516
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www.promega.com
TM248 · Revised 12/18
7.
Centrifuge briefly to bring the contents to the bottom of the tubes. Place reaction tubes on ice until ready for
thermal cycling.
8.
If using a thermal cycler that requires mineral oil, tilt the tubes and add one drop of oil to the side of the tube,
letting the oil run down the side of the tube.
7.D. Preparation of Agarose Gels
1.
Use NuSieve
®
3:1 agarose available from Cambrex.
2.
Choose a beaker 2–4 times the volume of the agarose solution.
3.
Add the appropriate amount of NuSieve
®
3:1 agarose and 1X TBE buffer to make a 4% gel (i.e., 100ml of 1X
TBE buffer and 4g NuSieve
®
3:1 agarose) to the beaker.
4.
Weigh the beaker, or mark the level of the liquid in the beaker.
5.
Mix to wet agarose.
6.
Soak the agarose for 15 minutes at room temperature.
7.
Heat the beaker in a microwave until the solution boils.
8.
Boil the agarose for 1 minute or until it is completely dissolved.
9.
Remove the agarose from the microwave.
10. Gently swirl the beaker to mix.
Caution:
Any microwaved solution may become superheated and boil over when agitated.
11. Add sufficient hot distilled water to obtain the initial weight or volume.
12. Mix thoroughly.
13. Cool the solution to 50–60°C.
14. Add ethidium bromide to 0.5µg/ml.
15. Pour the gel to a depth of 5mm. It is important that the gel is thin for optimum resolution.
16. Allow the gel to set.
17. Run gel in 1X TBE buffer containing 0.5µg/ml ethidium bromide. For best resolution, it is important ethidium
bromide is in both the running buffer and the gel.
18. Load samples.
19. Run gel at 5V/cm until the bromophenol blue dye front migrates to the bottom of the gel.
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