Recipes for miscellaneous salts, solutions, etc. (Khokha lab)

1% Agarose Plates (for use with trops or laevis)

1 g agarose powder + 100 ml 1/9 X MR without gent in 500 ml jar

   make no more than 300 ml (to prevent boil over)

Microwave on high 2 min/100 ml (w/ loose lid)

Swirl and check for solids

When completely melted, fill petri dish 1/4 full

Put in 4C for 5 min, or air dry 30 min

Lift out with tweezers

   -Can use a mold in agar before it hardens, or cut out desired shape after hardens

Bleaching solution (Sive et al. 2000, Mayor et al. 1995)

1% H202 5% formamide 0.5 X SSC(standard saline citrate) 4% BMB Blocking Reagent in MAB

Dissolve 10g BMB in 100 ml 1X MAB or 50g BMB (the whole bottle) in 500 ml 1X MAB(see above for MAB).

Make 50 ml aliquots in Falcon tubes. Store at -20C.

Bouin’s Fixative

For 50 ml: 35 ml Piric acid 12.5 ml Formaldehyde 2.5 ml glacial acetic acid Adjust volume, store at RT.

Cysteine, 2.0%

2 g cysteine in 100 ml 1/9 X MR

   pH will be around 2.0

Add stir bar and 4 NaOH pellets, stir until dissolved

Test pH, titrate to final pH 7.8 - 8.0 with 10 M NaOH

   cysteine will turn a faint purple as it approaches pH 7.8
   use HCL to titrate back if you go over

Dehydrating embryos

Embryos can be placed directly into 100% methanol in small glass vials.

dNTPs 10 mM

dNTPs (Roche), 100 mM (250 ul each)

Mix 250 µl each dNTP = 1000µl Add 1500µl ddH20 for final 2500µl of 10 mM solution.

Make 25-50 ul aliquots in eppendorf tubes. Store at -20C.

1M MgCl2

For 500 ml:

Dissolve 47.606 g MgCl2 in dH2O. Adjust volume, store at RT. 1M Tris pH 9.5

For 1 L:

Dissolve 121.14 g Tris in 700 ml dH2O. Adjust pH to 9.5 with HCl Adjust volume, store at RT.

Ficoll, 2.5%

2.5 g ficoll/100ml DI water (usually make 500 ml)

Autoclave and store at RT.

Use in place of water in dish to stabilize embryos 2.5% Ficoll in 1/3X MR (for use with X. laevis embryos)

Dissolve 2.5 g/100ml of Ficoll in 1/3X MR and autoclave(Takes long time to dissolve.)

Store at RT.

3% Ficoll in 1/9XMR (for use with X.tropicalis embryos)

Dissolve 3 g/100ml of Ficoll in 1/9X MR and autoclave(Takes long time to dissolve.)

Store at RT

Gentamicin Stock, Sigma (1000X, 100 mg/ml)

For 50 ml 1000X stock (50mg/ml):

Dissolve 2.5 g in 40 ml dH2O, adjust volume, filter sterilize with syringe and 0.22 uM filters to sterile falcon tube. Store at 4C.

Hybridization Buffer

For 4 L bottle: 2 L Formamide 1 L 20x SSC 4 g Torula RNA 0.4 g Heparin 40 ml Denhardt’s solution 4 ml Tween 20 4 g CHAPS Dissolve in dH2O, adjust volume. Store at -20C. Denhardt’s solution, 100X

For 100 ml 100X: BSA 2 g PVP-40 2 g Ficoll 2 g

Add a small amount of water to ingredients, make a slurry, then dilute. Store at -20C. Triethanolamine 0.1 M (pH7.0-8.0)

For 1 L: Dissolve 18.6 g triethanolamine in DEPC water, adjust pH with 8 pellets NaOH. Check pH with drops on pH paper. Adjust volume, store at RT.

Levamisol 1 M

Dissolve Levamisol (204.292 g/L) in dH2O, adjust volume, store at -20C. Alkaline Phosphatase (AP) Buffer, with Levamisol and Tween

For 1 L: 100 ml 1 M Tris pH 9.5 50 ml 1 M MgCl2 25 ml 4 M NaCl 1 ml Tween-20 2 ml 1 M Levamisol

Adjust volume, store in 50 ml aliquots at -20C.

MAB, 10X

For 4 L bottle: Maleic acid 464.4 g 350.64 NaCl

Start dissolving by adding around 280-320 g or more NaOH pellets. Add more pellets during the day as they dissolve. Procedure takes all day. Adjust pH to 7.5. Adjust volume. Store at RT.

MEMFA* (Sive et al. 2000)

 	1X 	10X 	for 1 L 10X

MOPS (pH 7.4) 0.1 M 1.0 M 209.27 g EGTA 2 mM 20 mM 7.608 g MgSO4 1 mM 10 mM 2.46 g Formaldehyde 3.7%

• Prepare a 10 X solution without the formaldehyde for storage. Add fresh formaldehyde(1/10 volume of standard 30% stock) prior to use. The solution will turn yellow with age and when autoclaved.

Paraformaldehyde(PFA), 20%

Measure 4g of Paraformaldehyde in the hood then add to 100 mls of pre-heated PBS.

Heat to around 62-64C.

Once dissolved aliquot 20 mls per tube and store at -20C.

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 4M NaCl, 1 liter

Dissolve 233.76 g NaCl in 750 ml dH2O. Adjust volume. HEPES 1 M, pH 7.4

For 1Liter: Hepes 238.31 g

Dissolve, Adjust pH and volume. Store at RT. TAE 50X

For 2 L: Tris 484 g 114.2 ml Glacial Acetic acid 200 ml 0.5 M EDTA pH 8.0

Dissolve in dH2O, adjust volume. Store at RT. 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 Sodium Phosphate Buffer(1M)

71 g Na2Hpo4 4 ml H3P04 1 Liter H20 titrate to pH 7.4

Modified Ringers

	1X	Mol. Wt.	10X	for 1L 10X*	For 10L 10X	For 20L 10X
NaCl	0.1M	58.44	1 M	58.44 g	584.4 g	1168.8 g
KCL	1.8 mM	74.56	18 mM	1.34 g	13.4 g	26.8 g
MgCL2	1.0 mM	203.31	10 mM	2.03 g	20.3 g	40.6 g
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

Phosphate Buffered Saline(PBS), 10X

10X For 4 L bottle: NaCl 320 g KCl 8 g Na2HPO4 57.6 g KH2PO4 9.6 g Dissolve in dH2O. Adjust pH to 7.4, and bring to volume. Store at RT.

Proteinase K(PK), 10 mg/ml

Supplied as 100 mg powder, (American Bioanalytical AB00925-000100)

Add 10 ml ddH20 for conc. of 10 mg/ml, mix until dissolved. Divide into 150 µl aliquots, store in -20C

PTw

1X PBS(phosphate buffered saline)

0.1% Tween-20

Rehydrating Embryos(for embryos stored in 100% methanol)

Draw off methanol with vacuum

Rehydrate embryos stepwise through each wash, 5 minutes each, on rotating rack:

3:1 methanol:PBS

1:1 methanol:PBS

1:3 methanol:PBS

100% PBS, repeat 2 - 3 times

RNase A (Sigma R 5000)

Dissolve 10mg/ml in TE (pH 7.8) and boil for 10 min before use. Store aliquots at -20C.

RNase T1 (Sigma R8251)

Dissolve 10,000 units/ ml (1000X) in 0.1 M sodium acetate at pH 5.5.

Boil for 10 min before use.

Store aliquots at -20C. While in use keep at 4C, avoid repeated thawing and freezing.

SDS 10%

For 1 L:

Dissolve 100 g SDS in 800 ml of dH2O. Adjust volume, store at RT. SSC, 20X

For 4 L bottle: NaCl 701.2 g Sodium Citrate 352.8 g

Dissolve in 3.3 L dH2O, adjust pH 7.0, and volume. Store at RT.