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INFORMATION FOR

Mapping

Protocol for Preparing 4% 50:50 Regular Agarose:NuSieve Agarose for SNP detection

protocol in Word format


1. Set up tray and combs in room temperature area. We use a multi-channel pipettor (Pipet-Lite LTS 8-channel pipette 2-20 uL, Model # L-8 20, Rainin) and the “DNA Plus Extra Wide Mini-Gel System with 23cm Width x 14cm Length UVT Gel Tray” made by Owl (purchased through USA Scientific), with 4 combs in the tray. This will allow you to run out two 96 well PCR plates at once.

a. Level tray and raise combs up by inserting pipette tips in the comb slots on the get tray, to prevent holes in bottom of wells.

i. We find pouring the hot agarose causes the tray to bow up, causing the comb to go too deep, piercing the bottom of the gel. Raise the comps up a bit with pipette tips to avoid this.

2. Add 250 ml 1/2X TAE to 500 ml beaker.

3. Weigh out 5 grams regular agarose and 5 grams NuSieve GTG Agarose(Lonza, #50084), total 10 grams.

Note:

NuSieve Agarose is very expensive, so handle with care.

4. Using stir bar in 1/10X TE and mixing plate set to 600, slowly sprinkle in agarose until it is all homogenized. Cover mixture with Saran Wrap with small vent hole pierced in top.

5. Chill mixture in 4 degree for 15 min.

6. Heat in microwave on Power 6 for 2 minutes, swirl.

7. Heat in microwave on Power 7 for 1 minute, swirl.

8. Set microwave to 5 minutes at Power 7. Continue to heat, stopping every 30 seconds to swirl. Be sure to carefully monitor for foaming, especially during first few minutes.

a. When foaming is evident, stop heating and swirl until foaming recedes. You may need to stop heating every 10 – 15 seconds during foaming, to prevent boiling over. After a few minutes, mixture will begin simply boiling, with no foam evident.

9. Solution is ready when no unmelted agarose is evident on swirling. This normally occurs after the 5 minute heating cycle in Step 8.

10. When solution is melted, quickly add 3 drops of 0.1 % Ethidium bromide, and stir solution on plate for 10 -20 seconds with stir plate on setting 400.

a. Setting higher than 400 may result in bubbles in agar.
11. Immediately pour gel, being careful to prevent stir bar falling in gel using a metal spatula.

a. Gel will begin to set rapidly, so pouring quickly is important.

12. Allow to cure for 1.5 – 2 hours before removing combs.

13. For SNP analysis, use 100 BP ladder (4 – 5 ul), and run gel at 120v for 45 minutes to 1 hour.

See related protocols:

Enzyme Selection and Primer Design for SNP detection

PCR and Digest for SNP detection