Agarose Gel 10X TBE Electrophoresis Running Buffer Ethanol Precipitation of PCR products Loading Dyes Queens Lysis Buffer RNA Storage and Stabilizing Solution

Agarose Gel

For 1% Agarose, dissolve 1g agarose into 100 ml 0.5X TBE buffer

For 1.5 % Agarose, dissolve 1.5g agarose into 100 ml 0.5X TBE buffer

Place Erlenmeyer flask containing agarose in solution into microwave for 1 minute, swirl, then microwave again until boiled for at least 10 seconds. Allow the solution to cool for one to two minutes before loading ethidium bromide (0.5 µg/ml).

10X TBE Electrophoresis Running Buffer

108.0 g    Tris base
55.0 g     Boric acid
9.3 g    Na₂EDTA

Add ddH2O to 1 liter.

Adjust pH to 8.3 using either NaOH (more basic) or HCl (more acidic).

Dilute to 0.5X for running standard agarose electrophoresis gels. 

Ethanol Precipitation of 15 µl PCR and other DNA Products

1. Add 1.5 µl 3M Sodium Acetate (for a 20 µl reaction add 2 µl, for 100 µl add 10 µl, and so on).
2. Add 60 µl -20⁰C 95% ethanol (for a 20 µl reaction add 40 µl, for 100 µl add 200 µl, and so on)
3. Vortex
4. Precipitate @ -20⁰C for 15 minutes.
5. Centrifuge @ 12,000 RPM for 30 minutes.
6. Pour off supernatant
7. Add 200 µl of -20⁰C 70% ethanol
8. Centrifuge @ 12,000 (or max) RPM for 10 minutes
9. Pipette off the supernatant, being careful not to scape the bottom of the tube but effectively drawing off the ethanol. A white pellet is usually noticeable at the bottom. You should not see a pool of ethanol in the boot of the tube, otherwise the sample will not dry properly. 
10. Air dry pellet at room temperature (approx. 30 min), preferably under a fumehood.
11. Resuspend in 20 µl 10mM Tris (pH 8.5) or ddH₂O
12. Check the concentration of your sample with the Nanodrop or on a 1% agarose gel using a concentration ladder.

 

Loading Dyes

When making these loading dyes you will notice that the glycerol is difficult to measure because it is like molasses. Use the graduated marks on the side of your tube to measure and cut the bore of the tip to effectively siphon the glycerol. Vortex after mixing and store at 4⁰C.

1. Bromophenol Blue
   
   1. EDTA 50 mM
   2. Glycerol 30% (w/v)
   3. Bromophenol Blue 0.5% (w/v)




2. Xylene Cyanol
   
1. EDTA 50 mM
2. Glycerol 30% (w/v)
3. Xylene Cyanol 0.5% (w/v)




3. Bromophenol Blue Xylene Cyanol Mix
   
1. EDTA 50 mM
2. Glycerol 30% (w/v)
3. Bromophenol Blue 0.25% (w/v)
4. Xylene Cyanol 0.25% (w/v)

Queens Lysis Buffer 1X

0.01 M Tris
0.01 M NaCl

0.01 M disodium-EDTA

1.0% n-lauroylsarcosine

pH 8.0

To make 100ml of 10X Queens Lysis Buffer:

1.21g Tris

0.48g NaCl

3.73g K₂EDTA

1g N-Lauryl sarcosyl

RNA Storage and Stabilizing Solution

Stock solutions and reagents required:

• 0.5 M EDTA disodium, dihydrate (18.61 g/100 ml, pH to 8.0 with NaOH while stirring)
• 1M Sodium Citrate trisodium salt, dihydrate (29.4 g/100 ml, stir to dissolve)
• Ammonium Sulfate, powdered; Sterile water.

Combine:

• 40 ml 0.5 M EDTA
• 25 ml 1M Sodium Citrate
• 700 g Ammonium Sulfate
• 935 ml of sterile distilled water

Stir ingredients on a hot plate under low heat until the Ammonium Sulfate is completely dissolved. Allow to cool, adjust the pH of the solution to pH5.2 using 1M H₂SO₄. Store either at room temperature or refrigerated.