imported>Virgilio at 21:15, 12 December 2011

2011-12-12T21:15:32Z

← Older revision		Revision as of 21:15, 12 December 2011
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	Prepare the final MBS solution by missing 100 ml of 10X salt solution with 7 ml of 0.1M CaCl2; adjust the volume up to 1 liter with distilled water. The following are the final concentrations:		Prepare the final MBS solution by missing 100 ml of 10X salt solution with 7 ml of 0.1M CaCl2; adjust the volume up to 1 liter with distilled water. The following are the final concentrations:

	: 88 mM NaCl		:88 mM NaCl
	: 1 mM CaCl2		:1 mM CaCl2
	:1 mP MgSO4		:1 mP MgSO4
	5 mM HEPES (pH 7.8)		:5 mM HEPES (pH 7.8)
	:2.5 mM NaHCO3		:2.5 mM NaHCO3

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	'''High-salt MBS'''		'''High-salt MBS'''

	: 0.1 M Cacl2 - 7 ml		:0.1 M Cacl2 - 7 ml
	:10x MBS salts - 100 ml		:10x MBS salts - 100 ml
	:5 M NaCl - 4 ml		:5 M NaCl - 4 ml
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	:2.0 mM Cacl2		:2.0 mM Cacl2
	:1.0 MgCl2		:1.0 MgCl2
	5.0 mM HEPES-NaOH (pH 7.6) or 300 mg/liter NaHCO3		:5.0 mM HEPES-NaOH (pH 7.6) or 300 mg/liter NaHCO3

imported>Virgilio at 20:28, 12 December 2011

2011-12-12T20:28:53Z

← Older revision		Revision as of 20:28, 12 December 2011
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			'''In this section:'''

			:Modified Barth's Saline (MBS)
			:Anitbiotics
			::Gentamycin
			::Oxytetracycline
			::Penicillin
			::Streptomycin Sulfate
			:High-salt MBS
			:Amphibian Ringers
			:Modified Frog Ringers (MR)
			:Marc's Modified Ringer's (MMR)
			:Normal Amphibian Medium (NAM)
			:Holtfreter's Solution


	'''Modified Barth's Saline (MBS)'''		'''Modified Barth's Saline (MBS)'''

imported>Virgilio at 15:16, 12 December 2011

2011-12-12T15:16:57Z

← Older revision		Revision as of 15:16, 12 December 2011
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	~~Culture Media~~


	'''Modified Barth's Saline (MBS)'''		'''Modified Barth's Saline (MBS)'''

imported>Virgilio: Created page with "Culture Media '''Modified Barth's Saline (MBS)''' Prepare two solutions: 0.1M CaCl2 and 10X MPS salts (Nacl/KCl/MgSO4/HEPES/NaHCO3) 0.1M Cacl2 :11.1 g/liter :autoclave and st..."

2011-12-12T15:16:29Z

Created page with "Culture Media '''Modified Barth's Saline (MBS)''' Prepare two solutions: 0.1M CaCl2 and 10X MPS salts (Nacl/KCl/MgSO4/HEPES/NaHCO3) 0.1M Cacl2 :11.1 g/liter :autoclave and st..."

New page

Culture Media

'''Modified Barth's Saline (MBS)'''

Prepare two solutions: 0.1M CaCl2 and 10X MPS salts (Nacl/KCl/MgSO4/HEPES/NaHCO3)

0.1M Cacl2
:11.1 g/liter
:autoclave and store aliquots at -20 deg C or 4 deg C

10X MBS salts
:880 mM NaCl
:10 mM KCl
:50 mM HEPES (pH 7.8)
:25 mM NaHCO3

Adjust final pH to 7.8 with NaOH; autoclave.

Prepare the final MBS solution by missing 100 ml of 10X salt solution with 7 ml of 0.1M CaCl2; adjust the volume up to 1 liter with distilled water. The following are the final concentrations:

: 88 mM NaCl
: 1 mM CaCl2
:1 mP MgSO4
5 mM HEPES (pH 7.8)
:2.5 mM NaHCO3

Note: Omit HEPES if MBS is to be used for oocyte maturation.

'''Anitbiotics'''

Antibiotics can be added to MBS. The following are the most commonly used antibiotics for embryos and explants.]

:''' Gentamycin''' (50 ug/ml)
::Make a 1000x stroke solution, 50 mg/ml, in water, filter sterilize and refrigerate

:'''Oxytetracycline''' (12.5 ug/ml)
:: Make a 1000x stock solution, 12.5 mg/ml, in water, Store frozen.

:'''Penicillin''' (commonly used for oocyte culture at 100 units/ml)
:: Make a 1000x stock stock solution, 100,000 units/ml, in water, Store frozen.

:'''Streptomycin Sulfate''' (Life Technologies)
::Make a 1000x stock solution, 100 mg/ml, in water. Store frozen.

'''High-salt MBS'''

: 0.1 M Cacl2 - 7 ml
:10x MBS salts - 100 ml
:5 M NaCl - 4 ml
:water - 888 ml

'''Amphibian Ringers'''

'''Amphibian ringers, 5 gal (19 L)'''

:NaCl 125.4 g
:KCL 2.85g
:CaCL2 2.85 g
:NaHCO3 3.8g
:DI water to 19 L

'''Modified Frog Ringers (MR)'''

:0.1 M NaCl
:1.8 mM KCl
:2.0 mM Cacl2
:1.0 MgCl2
5.0 mM HEPES-NaOH (pH 7.6) or 300 mg/liter NaHCO3

'''Modified Ringers'''

{|border="1" cellpadding="5" cellspacing="0" align="center"
|-
|
! scope="col" style="background:#efefef;" | 1X
! scope="col" style="background:#efefef;" | Mol. Wt.
! scope="col" style="background:#efefef;" | 10X
! scope="col" style="background:#efefef;" | for 1L 10X*
! scope="col" style="background:#efefef;" | For 10L 10X
! scope="col" style="background:#efefef;" | For 20L 10X
|-
! scope="row" style="background:#efefef;" | NaCl
|0.1M
|58.44
|1 M
|58.44 g
|584.4 g
|1168.8 g
|-
! scope="row" style="background:#efefef;" | KCL
|1.8 mM
|74.56
|18 mM
|1.34 g
|13.4 g
|26.8 g
|-
! scope="row" style="background:#efefef;" | MgCL2
|1.0 mM
|203.31
|10 mM
|2.03 g
|20.3 g
|40.6 g
|-
! scope="row" style="background:#efefef;" | Hepes
|5.0 mM
|238.31
|50 mM
|11.92 g
|119.2 g
|238.4 g
|}

*Add 100 ml of 10X to 1 L DI water for 1 L 1X solution

100 ml 1x MR

:2.5 ml 4 M NaCl
:1 ml 100x MR salts
:0.5 ml 1 M Hepes pH 7.6

'''100X MR salts'''

For 1L 100X:
:KCl 13.42 g (180 mM)
:CaCl2 29.4 g (200 mM)
:MgCl2 20.33 g (100 mM)
:Dissolve in dH2O, adjust volume. Store at RT.

'''1/3X Modified Ringers'''

For 20 L tank:
:NaCl 39 g
:66.7 ml 100X MR salts
:33 ml HEPES 1M pH 7.4

Dissolve in dH2O, adjust volume. Store at RT.

'''1/9X MR + 1% Gentamicin'''

300ml 1/3 X MR + 600 ml DI water

Add 900ul 1000X gentamicin stock

'''1/20X MR'''

100 cc 1/3 X MR 500 ml DI Water

'''Marc's Modified Ringer's (MMR)''' (Ubbels et al. 1983)

:0.1 M NaCl
:2.0 mM KCl
:1 mM MgSO4
:2 mM CaCl2
:5 mM HEPES (pH 7.8)
:0.1 mM EDTA

Note: Most current formulations of MMR omit EDTA and are adjusted to pH 7.4.

'''Normal Amphibian Medium (NAM)''' (Slack and Forman 1980)

:110 mM NaCl
:2 mM KCl
:1 mM Ca(NO3)2
:1 mM MgSO4
:0.1 mM EDTA
:1 mM NaHCO3
:2 mM sodium phosphate pH 7.4

Note: Assemble this solution from autoclaved stocks of 50x phosphate buffer (0.1 M sodium phosphate, pH 7.5) and a 10x stock of remaining salts excluding sodium bicarbonate. A 100x stock of sodium bicarbonate (0.1 M) should be filter-sterilized.

'''Holtfreter's Solution''' (Holtfreter 1931)

Prepare a stock of 25 mM NaHCO3 and 10x Holtfreter's salts. Autoclave the salts, and filter-sterilize the NaHCO3.

25 mM NaHCO3
:2.1 g/liter

10x Holtfreter's salts
:600 NaCl
:6 KCl
:9 mM CaCl2

Prepare the final solution by mixing 100 ml of 25 mM NaHCO3 with 100 ml of 10x Holtfreter's salts and adjust the volume of 1 liter with distilled water.

Culture media - Revision history

imported>Virgilio at 21:15, 12 December 2011

imported>Virgilio at 20:28, 12 December 2011

imported>Virgilio at 15:16, 12 December 2011

imported>Virgilio: Created page with "Culture Media '''Modified Barth's Saline (MBS)''' Prepare two solutions: 0.1M CaCl2 and 10X MPS salts (Nacl/KCl/MgSO4/HEPES/NaHCO3) 0.1M Cacl2 :11.1 g/liter :autoclave and st..."