Silver: Time-Lapse Microscopy - Revision history

Reshma P. Shetty at 18:11, 9 May 2007

2007-05-09T18:11:23Z

←Older revision		Revision as of 18:11, 9 May 2007
Line 57:		Line 57:
	[[Category:Protocol]]		[[Category:Protocol]]
	[[Category:Microscopy]]		[[Category:Microscopy]]
		+	[[Category:Yeast]]
		+	[[Category:In vivo]]

Reshma P. Shetty at 17:53, 9 May 2007

2007-05-09T17:53:03Z

←Older revision		Revision as of 17:53, 9 May 2007
Line 54:		Line 54:

	(Sheff MA, Thorn KS. Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae. '''Yeast''' 2004; 21: 661–670.)		(Sheff MA, Thorn KS. Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae. '''Yeast''' 2004; 21: 661–670.)
		+
		+	[[Category:Protocol]]
		+	[[Category:Microscopy]]

BrunoAfonso: /* Pad Preparation */

2006-08-15T00:35:56Z

Pad Preparation

←Older revision		Revision as of 00:35, 15 August 2006
Line 7:		Line 7:
	==Pad Preparation==		==Pad Preparation==

-	1. Microwave 2% agarose (mix of low-melt and normal, to taste) in Thorn media (see below).	+	1. Microwave 2% agarose (mix of low-melt and normal, to taste) in Thorn media (see below). (If you have used different percentages of agarose pads please let us know, bruno)

	2. Apply 2mL to a 24x60 mm coverslip.		2. Apply 2mL to a 24x60 mm coverslip.

BrunoAfonso: /* Pad Preparation */

2006-01-20T19:41:35Z

Pad Preparation

←Older revision		Revision as of 19:41, 20 January 2006
Line 11:		Line 11:
	2. Apply 2mL to a 24x60 mm coverslip.		2. Apply 2mL to a 24x60 mm coverslip.

-	3. Cover with additional coverslip, creating a sandwich. Let cool for 30 minutes.	+	3. Cover with additional coverslip, creating a sandwich. Let cool for 30 minutes. (1h seems to work better)

	==Cell Preparation==		==Cell Preparation==

JulianEskin at 21:07, 21 September 2005

2005-09-21T21:07:02Z

New page

Back to [[Protocols|OpenWetWare protocols]]

Back to [[Silver: Protocols|Silver Lab protocols]]

Back to [[Silver Lab]]

==Pad Preparation==

1. Microwave 2% agarose (mix of low-melt and normal, to taste) in Thorn media (see below).

2. Apply 2mL to a 24x60 mm coverslip.

3. Cover with additional coverslip, creating a sandwich. Let cool for 30 minutes.

==Cell Preparation==

4. Grow 5mL culture in Thorn media to OD 600 = 0.3. Back dilute if necessary.

5. Centrifuge at 2000 rpm for 2 minutes. Resuspend in 1mL Thorn media and transfer to a 1.5mL eppendorf tube.

6. Spin in a microfuge for 15 seconds to pellet. Resuspend in 100-200 µL Thorn media.

7. Once cooled cut a 5x5 mm square from the agarose pad. Apply 0.2 µL of cells to the top surface.

8. Invert square onto a glass-bottom imaging dish. Add a few drops of water to the dish (for moisture), and cover edges with parafilm.

9. Image.

==Thorn Media==

Standard media except with low-fluorescence yeast nitrogen base.

(Yeast nitrogen base without riboflavin and folic acid.)

* 5 g/l (NH4)2SO4
* 1 g/l KH2PO4
* 0.5 g/l MgSO4
* 0.1 g/l NaCl
* 0.1 g/l Ca2Cl
* 0.5 mg/l H3BO4
* 0.04 mg/l CuSO4
* 0.1 mg/l KI
* 0.2 mg/l FeCl3
* 0.4 mg/l MnSO4
* 0.2 mg/l Na2MoO4
* 0.4 mg/l ZnSO4
* 2 µg/l biotin
* 0.4 mg/l calcium pantothenate
* 2 mg/l inositol
* 0.4 mg/l niacin
* 0.2 mg/l PABA
* 0.4 mg/l pyridoxine HCl
* 0.4 mg/l thiamine

(Sheff MA, Thorn KS. Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae. '''Yeast''' 2004; 21: 661–670.)