1 Jul 2011


1. Make 350 mL of a 1% agarose gel as follows:  3.5 g agarose in 300mL ddH2O, stir briefly, cover beaker
With plastic wrap, and microwave on high for 5 min.  If agarose has not dissolve completely, microwave
An additional 1-2 min.
2. Transfer the beaker to a stirplate, remove plastic wrap, add 30mL 10X TBE (final = 1X TBE) and
20mL+ ddH2O (total volume of 300mL), and allow to cool while stirring.
3. Add 3 mL ethidium bromide solution while stirring, when agarose is <65°C.
4. Pour 50°C agarose into gel tray that has been wrapped and sealed with tape on both ends, and set up
With combs on a level surface.  Allow to cool to room temperature for a minimum of ½ hour, before
Attempting to remove combs.
5. Remove combs and tape from gel.  Place gel in gel box and cover with 1X TBE (~2L).
6. Remove 9 mL of cDNA from PCR tubes by carefully pepetting through the mineral oil layer, mix sample
Gently by pipetting up and down, wipe sides of pipet tip with a Kimwipe to remove excess oil, and
Dispense to a clean tube.  (NOTE:  Mineral oil does not need to be used with the 9600 PCR machine.)
7. Add 1mL loading buffer, mix by pipetting, and heat samples at 65°C for 6 minutes and 30 seconds. 
After heating, chill samples on ice (~2 min.), then microfuge briefly.
8. Load samples onto gel.  Run samples at ~120 V. Run time varies.  Usually, a 2-comb, 40-sample gel
Can be run in ~1-1½ hours.
9. After running the gel, nick a corner to define orientation of the gel.

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