Gattuso:RNA extraction - Revision history

Reshma P. Shetty: RNA extraction moved to Gattuso:RNA extraction: this is a lab-specific protocol, moving it into that namespace

2007-10-09T16:30:31Z

RNA extraction moved to Gattuso:RNA extraction: this is a lab-specific protocol, moving it into that namespace

←Older revision

Revision as of 16:30, 9 October 2007

Kerros at 17:18, 8 February 2007

2007-02-08T17:18:39Z

←Older revision		Revision as of 17:18, 8 February 2007
Line 22:		Line 22:
	2- Homogenize the sample using a seringe and needle (21G).		2- Homogenize the sample using a seringe and needle (21G).

-	3- Add 500 µl of extraction buffer and vortex for 30s. Vent to release any pressure built up.	+	3- Add 500 µl of extraction buffer* and vortex for 30s. Vent to release any pressure built up.

	4- Add 50 µl of 2 M sodium acetate, pH 4.0 and vortex for 30 s.		4- Add 50 µl of 2 M sodium acetate, pH 4.0 and vortex for 30 s.
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	10- Resuspend in 25 µl sterile water. Add an equal volume of 4 M LiCl and mix. Place on ice for 45 min.		10- Resuspend in 25 µl sterile water. Add an equal volume of 4 M LiCl and mix. Place on ice for 45 min.

-	11_ Centrifuge for 5 min at full speed. Wash the pellet in cold 70% ethanol, air dry and ~~rewsuspend~~ in small volume (50 µl-100 µl) of water	+	11_ Centrifuge for 5 min at full speed. Wash the pellet in cold 70% ethanol, air dry and resuspend in small volume (50 µl-100 µl) of water
		+
		+	*GITC buffer (RNA extraction buffer)
		+	4M guanidium isothiocyanate
		+	25 mM sodium citrate
		+	0.5% sarkosyl
		+	0.1% beta-mercaptoethanol
		+
		+	The buffer should be prepare using nanodrop water and autoclave before adding the beta-mercaptoethanol.

Kerros at 16:37, 5 February 2007

2007-02-05T16:37:51Z

Kerros: New page: Decalcification To 500 ml cultures of decalcifying cells add 11 ml of 0.1 M HCl to bring teh final concentration to 2.2 mM. Swirl the flask and note the clearing of teh culture. After ad...

2007-02-01T17:21:10Z

New page: Decalcification To 500 ml cultures of decalcifying cells add 11 ml of 0.1 M HCl to bring teh final concentration to 2.2 mM. Swirl the flask and note the clearing of teh culture. After ad...

New page

Decalcification

To 500 ml cultures of decalcifying cells add 11 ml of 0.1 M HCl to bring teh final concentration to 2.2 mM.
Swirl the flask and note the clearing of teh culture. After adding the HCl, check if teh pH has dropped from 8.0 to 5.0 using pH strips.
After 1 min, quikly neutralize the solution by adding 14.3 ml of 0.1 M NaOH (final concentration of 2.8 mM). This serves to restore the culture to a neutral pH of 8.0.

Haversting cells
Centrifuge at 10000g for 10 min at RT.
Do not place the cells on ice as this will cold shock the cells and activate specifics RNAses within the cell

1- if the RNA is to be extracted later:
Resuspend the cells in 100% ethanol and pellet the cells by centrifugation before storing at -20°C.

2- if teh RNA is to be extracted immediatly
Resuspend and combine the cells in a disposable centrifuge tube using centrifugation.

RNA extraction

1- Deep the tube in liquid nitrogen few minutes

2- Grind the cells into fine powder using a grinder adapted to the size of the tube.

3- Add 500 µl of extraction buffer and vortex for 30s. vent to release any pressure built up.

4- Add 50 µl of 2 M sodium acetate, pH 4.0 and vortex for 30 s.

5- Add 500 µl of water saturated phenol (pH 4.3) and vortex 30 s.

6- Add 100 µl of chloroform:isoamyl alcohol (24:1) and vortex for 30 s.

7- Centrifuge at room temperature for 10 min at 5000 g to precipitate protein and DNA.

8- Remove the RNA contained in the upper aqueous phase and add an equal volume of isopropanol. Mix and incubate at -20°C for at least 45 min.

9- Pellet teh RNA by centrifugation at 10000 g for 10 min at 4°C

10- Resuspend in 25 µl sterile water. Add an equal volume of 4 M LiCl and mix. Place on ice for 45 min.

11_ Centrifuge for 5 min at full speed. Wash the pellet in cold 70% ethanol, air dry and rewsuspend in small volume (50 µl-100 µl) of water

←Older revision		Revision as of 16:37, 5 February 2007
Line 1:		Line 1:
	Decalcification		Decalcification

-	To 500 ml cultures of decalcifying cells add 11 ml of 0.1 M HCl to bring ~~teh~~ final concentration to 2.2 mM.	+	To 500 ml cultures of decalcifying cells add 11 ml of 0.1 M HCl to bring the final concentration to 2.2 mM.
-	Swirl the flask and note the clearing of ~~teh~~ culture. After adding the HCl, check if ~~teh~~ pH has dropped from 8.0 to 5.0 using pH strips.	+	Swirl the flask and note the clearing of the culture. After adding the HCl, check if the pH has dropped from 8.0 to 5.0 using pH strips.
	After 1 min, quikly neutralize the solution by adding 14.3 ml of 0.1 M NaOH (final concentration of 2.8 mM). This serves to restore the culture to a neutral pH of 8.0.		After 1 min, quikly neutralize the solution by adding 14.3 ml of 0.1 M NaOH (final concentration of 2.8 mM). This serves to restore the culture to a neutral pH of 8.0.

Line 12:		Line 12:
	Resuspend the cells in 100% ethanol and pellet the cells by centrifugation before storing at -20°C.		Resuspend the cells in 100% ethanol and pellet the cells by centrifugation before storing at -20°C.

-	2- if ~~teh~~ RNA is to be extracted ~~immediatly~~	+	2- if the RNA is to be extracted immediately
	Resuspend and combine the cells in a disposable centrifuge tube using centrifugation.		Resuspend and combine the cells in a disposable centrifuge tube using centrifugation.

Line 20:		Line 20:
	1- Deep the tube in liquid nitrogen few minutes		1- Deep the tube in liquid nitrogen few minutes

-	2- ~~Grind~~ the ~~cells into fine powder~~ using a ~~grinder adapted to the size of the tube~~.	+	2- Homogenize the sample using a seringe and needle (21G).

-	3- Add 500 µl of extraction buffer and vortex for 30s. ~~vent~~ to release any pressure built up.	+	3- Add 500 µl of extraction buffer and vortex for 30s. Vent to release any pressure built up.

	4- Add 50 µl of 2 M sodium acetate, pH 4.0 and vortex for 30 s.		4- Add 50 µl of 2 M sodium acetate, pH 4.0 and vortex for 30 s.
Line 34:		Line 34:
	8- Remove the RNA contained in the upper aqueous phase and add an equal volume of isopropanol. Mix and incubate at -20°C for at least 45 min.		8- Remove the RNA contained in the upper aqueous phase and add an equal volume of isopropanol. Mix and incubate at -20°C for at least 45 min.

-	9- Pellet ~~teh~~ RNA by centrifugation at 10000 g for 10 min at 4°C	+	9- Pellet the RNA by centrifugation at 10000 g for 10 min at 4°C

	10- Resuspend in 25 µl sterile water. Add an equal volume of 4 M LiCl and mix. Place on ice for 45 min.		10- Resuspend in 25 µl sterile water. Add an equal volume of 4 M LiCl and mix. Place on ice for 45 min.

	11_ Centrifuge for 5 min at full speed. Wash the pellet in cold 70% ethanol, air dry and rewsuspend in small volume (50 µl-100 µl) of water		11_ Centrifuge for 5 min at full speed. Wash the pellet in cold 70% ethanol, air dry and rewsuspend in small volume (50 µl-100 µl) of water