Analysis of 384 PCR Products for Microarrays

Materials:

• Agarose, powder form………………………………………….….Reagent Room

• 10xTBE……………………………………………………………Above Gel Bench

• Ethidium Bromide solution………………………………………..4 Degree Refrigerator

• p10 tips for Finnipipette…………………………………………...EST Work Area

• 6x loading buffer…………………………………………………..-20 Freezer

• 96 well PCR plates…………………………………………………Under EST Work Area

• Hydra 96 Pipette Robot…………………………………………….EST Work Area

Reagents:

• TBE Working Solution: Mix up 3 Liters of 1xTBE, by diluting 300mls of 10xTBE into 2700mls of nanopure water.

• 1% gel solution: Mix together 5g of agarose with 500mls 1xTBE and heat as described in following protocol.

Protocol:

1. Set up the large agarose rig by setting the gel holder into the casting apparatus and closing tightly.

2. Place the 8 fifty tooth combs evenly throughout the gel holder and tighten into place. The final comb does not have a clamp; therefore it will need to be taped into place.

3. Mix together the 1% gel solution in a 500ml flask and place a paper towel in the mouth of the flask.

4. Heat the flask in the microwave for 3:30, watching constantly. If the mixture starts to boil, remove flask and gently swirl. Continue to heat until all agarose has been completely dissolved in the TBE.

5. Remove the 1% agarose solution from the microwave (remember it’s really hot!!!). Obtain 20ul of EtBr mix and add to the flask, swirl gently to mix. ****Ethidium Bromide is a Mutagen, you must wear gloves any time you are handling the solution, or the gel after it has been added.

6. Slowly pour the 1% argarose mixture with EtBr into the casting set up. Pour slowly to assure there are no leaks and that the expanding tray does not crack.

7. Allow the gel to solidify for 20-30 minutes.

8. Dispense 900ul of 6x loading buffer into a reagent reservoir.

9. Using the small multichannel pipette, dispense 2ul of loading buffer into each well of four 96 well PCR plates.

10. Turn on the hydra 96 and set on program #11. Perform one full bleach wash and one full water wash.

11. Change to hydra program #5 and place the first set of 96 PCR products into the machine. Fill the hydra and remove PCR plate. Dispense the products into the corresponding loading buffer plate.

12. Wash the hydra in program #5 and repeat with the remaining plates of PCR products.

13. Remove the combs from the casted gel and release the gel from the casting apparatus. Place the gel in the gel box and add the 3 Liters of 1xTBE.

14. Using the small multichannel pipette pick up the 7ul samples from column 1 of the first product/loading buffer plate. Dispense these samples in every other well of the first gel row (this is how the multichannel will line up).

15. Using new tips, load the second set of samples (column 2), so that they fill in the gaps between the first set. Continue this pattern until all 384 samples have been loaded.

16. Replace the lid of the gel box so that the red lead is at the bottom.

17. Run the gel at approximately 100V for 45 minutes.

18. Slide the gel off of the holder into a large tray and cut the gel in half using the large spatula.

19. Take a picture of each half of the gel using the 8^th floor gel camera and label each picture.

20. Analyze the gel and mark unamplified clones on data sheet.