高分子量RNA杂交

Allison C. Mallory, Bartel Lab Whitehead Institute

For 1% Agarose Gel:

Stock

1. Boil to dissolve agarose

2. Cool it to about 60℃

3. Add formaldehyde to a final concentration of 7% (18ml of lab 40% stock) and mix well and pour (I use the thin 1mm well comb).

4. Run gel in 1 X FF buffer with 0.7% formaldehyde (18ml of lab 40% stock in 1L of 1 X FF buffer)
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10 X FF running buffer:

Final concentration

200mM HEPES sodium salt

10mM EDTA

pH buffer to 7.8

For 500mL 10 X FF: add 23.8g HEPES and 1.9g EDTA to 500mL with water (don’t forget to pH to 7.8)
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To prepare RNA for loading:

I usually load 5-15ug of RNA (depending on the concentration of my RNAs and the abundance of the mRNA I am trying to detect).

Master Mix:

Stock

1. Calculate the volume of each RNA needed for Xug.

2. Add 12ul of Master Mix to each tube.

3. Add appropriate amount of RNA (the final volume of the sample, including master mix, should not exceed 16ul to be able to fit it into the well)

4. Fill each tube to a final volume of 16ul with water (final ratio of Master mix to RNA is 3 volumes to 1 volume)

5. Incubate at 85C for 2 minutes to denature RNA

6. Put RNA on ice and quickly spin down each sample (leave samples on ice while loading the gel

7. Run gel until BPB dye is about 3/4down the gel then take a picture.

Transfer to Nylon membrane (Genescreen Plus/NEN) by capillary action

1. Wash gel in water

2. Soak gel in 50mM NaOH, 10mM NaCl for 10 minutes at room temperature (with shaking)

3. Neutralize by shaking for 10 minutes in 2.5 X TBE at room temperature

4. Equilibrate the gel and the nylon membrane by soaking them in 10 X SSC at room temperature

5. Transfer RNA to nylon membrane in 10 X SSC overnight。

To make transfer sandwich:

1. Place a support wrapped with 3mm Watman paper in a dish containing 10 X SSC(the SSC should not wash over the support)

2. Place gel upside down on support

3. Place parafilm around the gel to ensure that buffer only transfers through the gel

4. Place the membrane on top of the gel

5. Place two pieces of 3mm Watman paper on top of the membrane

6. Roll out any glass bubbles with a glass rod

7. Place a stack of paper towels on top of the sandwich

8. Put a glass plate on top of this and place a 500mg weight on top of this

9. Let the RNA transfer overnight.

10. Next morning, remove paper towels and Watman

11. Place membrane RNA side up, UV X-link with 100uJ of energy, and bake at 80℃ for 30 minutes to dry the membrane.

Prehybridize, Hybridize, and wash membrane in Ambion Ultrahyb buffer following manufacturers protocol.

In vitro transcribed RNA probes(using Bartel lab T7 polymerase):

Incubate at 37C for 1hour

Random primed DNA probes:

I use Amersham Megaprime labeling kit following manufactures protocol.

Label with alpha-P32 dCTP (6000Ci/mmol) NEN cat#: BLU013Z