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Northern Blot

 

Day 1
(Morning)

 

0
(Safety)

 

1.   Use solutions prepared on DEPC-water only

2.   Do not touch anything with your hands! Treat the gloves with an RNase inhibitor (ex. Ambion RNaseZap, #9780 for $42.00)

3.   Use RNase free pipette tips (ex. Costar Corning filtered tips avail. from Fisher Scientific, #4809, #4821 and #4823 for $45.00 each)

 

I
(Preparations)

 

1.   Prepare the gel and the running buffer:

 

For the gel, mix:

 

Regular agarose

.96 g

Low melt agarose

1.44 g

Na2EDTA (.5M; pH 8.0)

.8 ml

Triethanolamine (1M; pH 7.0)

8 ml

Water

174 ml

 

Microwave the mixture until it becomes liquid (2-3 min in high mode).
In the hood, add SDS, 10% (1 ml) and 37% formaldehyde (16 ml). Mix it well and fill up a gel tray. Let it polymerize for 20 min.

 

For 2l of the running buffer:

 

Na2EDTA (.5M; pH 8.0)

9 ml

Triethanolamine (1M; pH 7.0)

90 ml

37% Formaldehyde

188 ml

Water

1713 ml

 

Mix it well.

 

2.   Prepare a gel tray and fill it up with the gel mixture. Let the gel to polymerize (20 min). The prepared gel mixture is enough to fill a 8 x 9.5” gel tray.

3.   Prepare the samples

 

For the sample buffer:

 

Na2EDTA (.5M; pH 8.0)

1.2 mcl

Triethanolamine (1M; pH 7.0)

12 mcl

37% Formaldehyde

50 mcl

Deionized formaldehyde solution

150 mcl

Water

1.1 mcl

Bromophenol blue (4 mg/ml) plus xylene cyanol (4 mg/ml) and FicollTM 400 (250 mg/ml)

85.7 mcl

 

To prepare samples, mix together: 20 mcl of the sample buffer and 20 mcg of the RNA. Add 2 mcl of ethidium bromide (optional). For RNA Mw markers you must add ethidium bromide. Incubate samples at 65oC for 10 min and chill them on ice for 5 min.

 

II
(Electrophoresis)

 

1.   Cover gel with the running buffer.

2.   Load samples on the gel

3.   Set up 70-80 V & 10-20 mA. Run the gel for 4 h

 

Enjoy your lunch!

 

III
(Washing)

 

1.   Wash gel from formaldehyde with 0.15 M NaCl (3 x 15 min)

2.   Wash gel with 20x SSC for 30 min. If your samples contain ethidium bromide, you can take a picture of your gel (optional)

For 1l of 20x SSC dissolve 88.23g of tri-sodium citrate and 175.35g NaCl in 800 ml of water. Adjust pH to 7-8. Add water to 1l, filter the solution and store it at room temperature for 3 months.

 

3.   During the last wash, prepare a membrane for transfer (ex. Amersham-Pharmacia Biotech, Hybond-N+ membrane #RPN203B, $239/roll). Cut membrane to appropriate size (1/4” bigger than the gel in each dimension), soak it in water (5 min) and wash in 20x SSC (15 min)

 

IV
(Installation of transfer tray)

 

1.   Fill up a transfer tray with 20x SSC

2.   Put a glass across the tray

3.   Prepare a bar of filter paper: make a paper bar, 0.5-1 feet length. Make the bar a little bit (~1”) wider than the gel, soak it in 20x SSC and put ends of the bar into the tray. Remove the bubbles from under the paper with a plastic pipete

 

V
(Assembling a sandwich)

 

1.   Put the gel on the filter paper bar, remove the bubbles under from the gel and wrap any space between the tray and the gel to improve the transfer efficiency

 

2.   Prepare some sheets of filter paper (a little bit wider than the gel in any dimension) and wet one of them in 20x SSC. Cover gel with the membrane and a wet sheet. Remove the bubbles and put the remained dry sheets on the top.

 

3.   Put 4-5“ of C-folded paper towels on the top of the sandwich and make a support on a back to prevent sandwich from collapsing. Very gently press the towels down with a weight of 1-1.5 lb. (two Sigma-Aldrich old catalog books is OK) and leave sandwich overnight for a transfer.

 

Have a good night!

 

Day 2
(Morning or early afternoon)

 

VI
(Crosslinking)

 

1.      Disassemble the sandwich and put the membrane under the UV-light for 1-2 min. If you have Stratagene UV Stratalinker 1800, #400671, use autocrosslink mode twice.

 

2.   Bake membrane in hybridization chamber (80oC; 2h)

3.   Take a picture of the gel (optional)

 

VII
(Prehybridization)

 

1.   Soak membrane in water for 5 min and 20xSSC for 10 min

2.   Fill up a hybridization bottle with 20x SSC and put membrane inside the bottle. Face it up. Carefully empty the bottle.

3.   Add 0.5-1 oz of hybridization buffer to the membrane and incubate it at 65oC for 2 h with slow rotation

 

VIII
(Hybridization)

 

1.   Prepare a probe and a separation column. In the lab, we use rediprime II random prime labeling system (Amersham-Pharmacia Biotech, #RPN1633, $237) to label the probe and MicroSpin G-50 columns (Amersham-Pharmacia Biotech, #27-5330-01, $152) to purify it.

2.   Prespin the column (750g; 15s) Load probe on the column, spin down (750g; 2 min) and save the filtrate

3.   Drop the filtrate into hybridization buffer and continue the incubation overnight

 

Have a good evening!

 

Day 3
(Morning or early afternoon)

 

IX
(Washing-2)

 

1.   Wash membrane with 2x SSC plus 0.5% SDS for 30 min at room temperature. I do it at a shacker in a tray at a high speed.

2.   During this wash, preheat double volume of 1x SSC plus .5% SSC to 65oC

3.   Double wash the membrane in the preheated buffer for 15 min

 

X
(Preparation of a cassette)

 

1.   Pick up the membrane and dry it briefly between two C-folded paper towels

2.   Cut plastic wrap and put it in a cassette. The piece of wrap should be 2-3 times wider than the cassette. Put the membrane in the cassette on the wrap film and cover it with the wrap.

 

3.   In the dark room, place a film on the wrapped membrane and start to exposure the film

 

Lunchtime...

 

XI
(Processing)

 

1.   Process the film in couple of hours. Based on the band intensity, estimate optimal time for the film exposure. If signal is too low, the exposure can take for couple of days.

 

2.   Expose membrane to another film for the estimated exposure time

 

3.   Strip membrane in boiled 0.5% SDS and reprobe with a housekeeping gene: boil SDS, take it off the heat and drop membrane with the face side down. “Striptise” will be over in about 30 min. Pick up the membrane and reprobe it with a housekeeping probe (ex. 28S cDNA)



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