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NSF Soybean Functional Genomics Project: Workshop: Electrophoresis of PCR Products


NSF Soybean Functional Genomics
Vodkin Laboratory, University of Illinois
NSF Home Overview Investigations

Workshop
Protocols

Soybean
EST Project

NSF Soybean Functional Genomics Project
Vodkin Laboratory, University of Illinois
Preparation of DNA for Microarrays, page 4, 5
Steve Clough/Reena Philip
Electrophoresis of PCR products with Life Technologies Sunrise gel apparatus
Gel:

In a 500 ml Pyrex« glass bottle, add:
Agarose:
3 g
H2O
270 mls
10X TA
30 mls

Screw cap partially on.
Microwave at high power for 2 minutes.
Carefully swirl (do little-by-little in case it boils violently) to mix in agarose.
If agarose is not fully dissolved, microwave another 20 - 30 sec, repeating until dissolved.
Cool until 55-60oC.
Pour into taped gel tray, add four combs (52-toothed).
When completely solidified (about 20-30 minutes) remove tape.
Place into gel box (electrodes in front, top row of wells on right).

Add 1X TA (about 1500 mls) until the buffer just covers the wells.

Samples:

Dilute 5X dye to 1X dye. (100 Ál of dye to 400 Ál of water).
Add 5ul of 1X dye to each well of a 96well plate.

Remove plates from the refrigerator. Vortex them for a few seconds and spin them down at 4000 rpm for 1 min.
Load 0.5 Ál of samples using Finnpette manual 8 channel, 0.5-10ul pipettor and finntips.
Cover the original sample plate with 4 inch wide tape and seal it well. Store plates in the refrigerator.

The plates containing the samples to be loaded on the gel should be sealed with a 3 inch wide tape. Spin them at 4000 rpm before loading on the gel.
Samples 1A-H will go into top right lanes (every other well is skipped).
Follow in numerical order so 1A-3H go across right row, then 4A-6H, 7A-9H, and 10A-12H across far left.

We use GIBCO Low DNA Mass Ladder (catalog no. 10068-013) at 200 Ál per vial.
Dilute 200Ál of the ladder with 800 Ál of 1X loading dye.

Add 10 ul of the diluted Low DNA Mass Markers to one of the remaining wells at end of each row (stagger so marker is put into well#49 for first row, well#50 in second, well #51 in third row and well #52 in last row).

Run:
Connect to power supply.
Run at 100V about 75 minutes.

Stain:
Cut gel in half such that cut is just at the 3rd set of wells.
Slide each half of gel off gel tray into separate pre-labelled staining trays, each containing about 500 ml of de-ionized water from the tap.
Put on shaker at slow speed (just above #2).
Slowly add 10 ul of Ethidium Bromide at10 mg / ml.
Leave shaking about 30 minutes.
Decant stain into a waste container and destain gel with water about 15 minutes.
View/photograph with Kodak gel documentation system.
Gel: Bright bands

 


* Department of Crop Sciences
* College of Agricultural, Consumer, and Environmental Sciences
* University of Illinois at Urbana-Champaign


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